141
TO WI% cvidenf~
hr
the ~~v~~lv~m~nl nf asffylchof;ncstcr;tsc~
activity in the ml~~uiaf~?~ in&cncc
nf the airwy
oxamincd responses to ~c~F~~c~~~~~~~ (ACh), ~~Fh~n~~h~~~,histamine or KCl in isolated cpjFh~fium-intact guinca-pig Frachcatis prcparaFi[)ns. The conccntralitln-response CUNCS to ACh wcrc shifted %-fold dcnudalion
but the contractile
rcspansc to KC1 was nut attcrcd. The rcsponsc tn histamine
increased 4-fold with no attenuation to ACh
in cpithclium-intact
in the prcscncc of physostigmine
Fissucs more (about
prcscncc of phys~~stigmioc rcmcwing the cpithciium contract the trachea. Removing ntrt a substrate olccfrical
field-srimulatcd
tissues
had only a slight cffcct (not statistically
preparations
(30 nM) potcntiated the response (abouF 3%foid).
Si~niri~~nF}
Thus,
in the
on the potency of ACh to
the epith~l~um had no cffcct on the potency of b~th~Incchol, a muscarinic receptor agonist that is
for eholincsttrascs.
rcspcrnscs in cpithclium-intact
Izb-fold) than in cpithclium-denuded
to the left by cpithclial
in cpith~:ium-dcnudcd
(30 nM). Physostigminc
~p~th~rium, we
and ~pithcli~m-denuded
Phys~?sFi~minc itself
contracted
and dcnudcd preparations
chnlincrgic
mccbanism by which the cpitbclium
contractions
were unaffected
inhibi+,; contraction
Fhc trachealis
wore not significantly by removing
muscle but the pD, diffcrcnt.
The
the epithclium.
tialues and maximum
frcqucncy-rcsponsc In conclusion.
cmvcs In
the principal
of guinea-pig trachea to exogcnously applied ACh is via epithclium-de-
rived acctylcholincstL;rascactivity.
1. Introduction While the cffccts of cpithclium removal and the protective role of cpithclium in airway hypcrrcsponsivencss have hcen investigated (Aizrrwa et a!.. I%%; ~unakata et at.. 19X9) and the possihlc existence of an ~~jth~lium-d~r~v~d retaxing factor (EpDRF) has heen suggested ~Ts~~~r~art et at,, 198x7; Fcrnandcs ct al.. 19891, the topic remains c~)ntr~versi~ll, Either attenuation (1R;irncsCF al., I!%+% ~iavah~ln et al., 198% or ffo attenuation in the potencies of some relaxants by cpithelium-removal (Lund&d and Persson, 1988) has hecn seen and the supcrscnsittvily may he due to removal ot a permeability barrier rather than to EpDRF (Nolrc~ydc, l%ft; Undem et al., I9XX; Small et al., 1990; Yang ct al., 1991). Removing the cpithciium from isolated guinea-pig trachea potcntiates the response to a variety cf contractile agonists such 3s
acstylcholinc (AChI (Barnes et al., 198% Nolwydc, 1986; lriarte et al., 1990; Small et at., :090). Interestingly, in contrast to the rcspunsc to AC%, contractions due to mcthacholinc, carkramylcholinc and hcthanechol are only slightly, if at all, potent&cd by epithelium removal (Goldic ct al., 1986 Hay cl al., I9Xh; Racburn et al,, 19fz(i;Smali et al., Iclf)iff, This sugggcsts a role for ~p~th~liurn-d~rjved enzymes in the rn~ta~~~l~srnnf cho~~ncr~~~agents since the order of ~~)t~fit~ation of the agents corrcsyonds to that of their sus~~~tib~~ityto !?ydrolysisby ;rcetylcholinestcrasc. The purpose of the prcscnt study was to dctcrminc if ~~itf~~liun~-deriv~d acctylcholincstsrasc arn SCTVC to limit ~~~~tra~ti~~ns due cxogcnous tcl ACh or to AC% rclcascd ~nd~g~nuusly via neuronal stirnn~~ltj(~n.
tively. To obtain quantitative EFS responses, stimulation was given appr~~xim~tciy once cvcry 5 min using a 10-s stimulus train Wndem et al., 1990).
ACb, bethanechoi, l~istamine and KC1 were administered cumulatively to a trachealis muscle preparation with denuded or intact e~ithelium. The tracheal ep~theiiunl was removed mechanically by gentle rubbing with a cotton swab. The absence of cpithei~~m was confirmed h~st~iog~callyas previously reported (Ffavahan et al.. 19%; Koga et al., 1989; Daffonchio et al., 1990). The acetylcholincstcrasc inhibitor, physostigmine, was used with both epithelium-intact and denuded preparations. The concentration utilized (30 nM1 for the physostigmine pretreatment study was the threshold concentration of physostigmine which elicited a c~)ntractiie response (see Results). For the ~iectr~c~l field stimulation (EFSI studies, propranoiol (3 FM) and indomethacin (3 PM) were included in the buffer soIution to avoid effects of @-receptor stimulatio!~ by catccholamincs and by prostaglandins, respectively, on the neuronal responses (lto and Tajima, 19X1). To confirm the cholinergic nature of the response to EFS. atr~~pine (1 FM) was added at the end of each EFS cxpcriment. Drugs were prepared and diluted in 0.9% NaCI, with the exception of ind~)methacin (in absolute ethanol at a stock concentration of 30 mM with subscquent dilutions), and wcrc added to the organ baths in increments of 0.2 ml or less. Atropine sulfate, acctyicholine chloride (AChf, bctha~cch~~~ hydr~h~or~de, histamine dihydr~~~hloride, ind~~methacin, physostig-
TABLE pD,
1 and concentralion
values
;rhscnce(Control)
ratin
for conlractilc
and presence of physostigmine
Agt.onist
agaiists
in epilhdium-intact
and epithrlium-drnuded
guinr;c-pig trachcnlis mu%&
in the
(30 nM).
C’ont rol
Physosti~mine-prctrcated
Epithelium
Ccmcentcltion
nh
Epithclium
rati0
Conccnfralion rati
-intact ”
-dmuded ”
ACh
4.94 * 0.11
6.25 + O.fth ‘
7.27 + 3.91
Bathanechol
5.71*
f?
S.h:! +tt.ofi
0.x.1 * 0.15
s.h.l+
WI6
S.hX * ll.tt.5
I.iXrf-Il.34
KCI
I .97 + 0.07
5
7.03 rt 0.0~
3.1O+tLO8
‘.Oh + 0.07
2. I I f 0.10
I. Ih + 11.34
3
Histamine
5.X _t- 0. IO
S.X’~O.10
4.‘-?*
1.1.5
4.97 + 0.
h.h.5+ I .w
h
uxpcrimenls
Ch n
”
pD,
= - log(EC5,,1.
Thr
0.115
values
’ P < 0.05 vs. epithrlium-intact that for ACh thrre
reproscnt
26.44 + 7.X
c mc;rns+S.E.
preparation without
was no significant
-intact -’
of
6.36+021
(St. Louis,
MO,
6.7Y*l1.13
IO
s.h7+0 _
indicates
c
. I I cd
the number
of tissue
strips)
in each paired group.
” P < 0.05 vs. epithelium-intact preparation with physostigmine i30 r&ii. epithelium-intact and denuded physostiSmine-pr~trratrd prcparalions.
t-test for paired data; P < (3.05 was cant.
considered
Note
signifi-
U.S.A.).
3. Intrinsic b~than~~hul
i‘
physostigmine.
difference betwern
mine salicylate. potassium chloride WCl>, dl-propranolol hydrochloride wcrc all obtained from Sigma Chemical
-denuded d
activitjj (76 of maximal response to 0.1 mM for contractile agonists studies)
and potency (assessed as negative log,,, molar EC,,,) of agonists were calculated from individual concentrationrcsponsc curves. Differences in sensitivity to the agonists under various conditions (without/with epithelium) were quantified by measurement of the concentratton rattos at the ECSo. Contractions obtained with clcctrical field stimulation (EFS) were nornlali~~d as percentages of the maximal response to EFS, taken as 100%. The values arc given as the means + S.E. Unless otherwise stated, the difference between two means was analysed for statistical significance by Student’s
Results
The concentration-response CUIVCS to ACh were shifted 70 the left (2h-fold) by epithelium removal (fig. 1A). In :%lc cpithclium-denuded preparations, the conccntration-rcsponsc curves in the presence of physostigmine (30 nM) were slightly (Xi-fold) shifted to the left (fig. 1A), ncvcrthclcss the pD, values in the absence and presence of physostigminc (30 nM) wcrc not significantly different (table Il. Both epi~hcfium-intact and cpitilclium-denuded prcp~~rat~:)ns prctrc~ltcd with physostigminc (50 nM) showed significant potcntiation (P < 0.05) compared to iI physostigmine-untrcatcd ithclium-intact
cp-
control prcparetion (fig. IA. tablo I).
log [KC11 W Fig. 2. lq! symlr)ls)
cuncentr;lti(,n-rcs~(~lls~ or ep;thcIium-dcnudccl
curves to KU (closed ~ymbls)
(A) :mcf hisl;uninu preparations
(B) in isdatcd
Ir;WlWdis
arc cxprcsscd ;tc pcrccnta~cs of the maximum rcspcmsc ta either KCI (A) or hist;tmint:
(A) and six fttr f~is~~irnin~ [R) ~xp~rim~~ls,r~sp~cti~,~l~~ (5~2 &I& physosti~millc the rasponsc
musck. Respnn~s
in the ahsencc (circle) or prcscnce ttri:m&)
If.
f
13) and art
bars indicate S.E.M.
sht\wn us means
with intact trlpcn (311nM). The &t;l
of follr cWrin)rnts filr Kf‘l
Ntttc th;t~ ncitbcr cpith~iium rcmtrvat 11~ cpitllrlit:itl-remItvsl pl~tcnli~~tclt prctrc~itn~~nt (30 nMf p(~tcnti~~tc~ thr rcsptmse to KC’I (upper 10 mMt (A). On the ttthcr had to histaminr (I’ < O.fiS~. while phyb0sti~mine pretreatmcnl W nM1 clitl n(lt incrc;*sc thf potency of ~pitl~cl~~~m-inl~~cffisWc. indicnting that histilminc was not Ity accrylcllolincslcrasc activity 111). ilffL%lCd
Vcrtbl
wcrc oht;lind
trf phy%l&!minc
treatment at low concentrations of KCI (below 10 mM) (figs. li3 and :A, tahlc 11. Concentration-res~nse curves to histamine were shifted to the left about 4-fold by epithet&m removal (fig. 2B, table 1). The cxtcnt of the ieftward shift caused by ~pithclium removal was not influcnccd by pretreatment with phys~~stigmine(3t(t nM) (fig. 2B, table 1). The intrinsic activities i% of the maximum responses to bcthancchol t1.t mM1 for ACh, histamine and KCI were 99 4 I, 98 + 2 and 96 + 3% (n = 41, respectively, and were not significantly d~ff~r~~t~ Fhysostigminc fro nM-16 J.LM) caused concentration-related contraction, followed by a smali decrease in the contraction (30 FM) (fig. 31. Thcrc were no significant diffcrcnces in either pD, values or maximum responses between epithelium-denuded and epithc~ium-intact preparations (table 2). Electrical field stimulation (EFSI (20 V, 0.1 ms, 10 s strains) resulted in a rapid c~?ntracti~~nthat reached a peak at 10 s. then dcclincd t~~wardbaseline on removal of the stimufus. In some preparations this rapid contra~ti~~n was f~~ll~~wedby either a slowly developing sccnnd contracti~~n or relaxation. The rapid first contraction was abolished by administ~tion of atropinc f f p!vlf (fig. 41. We quantized only the rapid atropinesensitive c~~ntract~onst% of maximum response) by increasing frcqucncy CHz) (fig. 51. The frequcncy-response curves to EFS in epitheljum-intact and in dcnudcd tissues were not signifi~ntly different (fig. 5, talk 2). Thus, in contrast to the rcsponsc to cxogc-
The concc~tr3tion-response curves to bethanechol and KCi were not sigl~i~~nt~y affected by ep~thelium ~rnoval or pbysostigmine pretreatment (30 nM) at the EC,, lev&, attho~gh a siisht feftward shift was obscrvtd uita cpithetium-rornn~ and ~hys~~sti~rnineprc-
Smin I-
I
L
I
0 1
0.3
1 0
A 2.0
I
L
I
I
h
A
0.0
8.0
16
32
64
!?OfHz)
i Atropine
I lpM
6dfHrf
f%. 4. ~~pr~s~nt~ti~~ ~~~i~l~~~r~phtrzrcing illustrnti~~ the r~sp(~nscsto cleotricaf field stjmul~tion (EFS) by incr~~~n~ frequ~t~~i~~ (Hz). The ~timuf~ti~~nwas given apprcwim:rtcly once cvcry 5 min using IfI+ trains. 2ttC’s intensity and (J.t-ms p&c duration. The contracliie rcsponsc was ctmfirmrd to havvehwn totatJy hlrtckcd tq ~dministr~tinn of nt~~)~in~(I _tiMt at the end of each experiment.
o-l
.Ol
.l
1
10
100
Frequency (Hz) Fig. 5. Lng frequent-res~nse curves to elect&d field stimulation (EFSI in is&red trachealis muscle. Responses were obtained in intact (open circles) or epithelium-denuded (closed circles)prepartilions. The data are expressed as percentages of the maximum responsc to EFS-induced contraction and are shown as means of seven experiments (see table 3. Vertical bars indicate S.E.M. There were no signi~cant differences in either potency or maximum response to EFS between epithelium:intact and epitheliom-denuded preparathns.
nously applied ACh, epithelium removal had no effect on the choiinergic contractions elicited by endogenous ACh.
4. Discussion The main findings of the present study were that epithciium removal in an open segment of guinea-pig trachea potcntiates the response to ACh about 26-fold and that this potentiation is almost entirely abolished by physostigmine pretreatment. Moreover, epitheiium removal had virtually no effect on the response to bethanechoi, a muscarinic receptor agonist that is not mctaboiized by ace~lchoiinesterase. These results indicate that the tracheal cpithclium inhibits contractions to exogenousiy applied ACh by metabolizing the agonist rather than by releasing a putative relaxant factor (Tschirhart et al., 1987; Fernandes et al., 1989). Our data showing that the choiinergic contractions in response to EFS and to physostigmine itself were unaffected by removal of the cpitheiium suggest that epithelium-derived choiinesterase activity does not serve to limit the smooth muscle response to the ACh released from parasympathetic nerve endings. The hypothesis that epithelium-derived acetyicholinesterase activity limits contractions to exogenousiy applied ACh agrees with previous reports that the relative degree of potentiation of cholinergic contraction produced by removing the epithelium was correlated with the susceptibility of the agonist to hydroly-
sis by acetylchoiinesterase (Goldie et al,, 1986; Hay et al., 1986; Smail et al., 1990). Acetic esters of cbolinc are considered to be susceptible to hydrolysis by analogy to the carbamic acid esters being resistant to hydrolysis Woelie, 1965). Thus, as a whole, the order of ~tentiation caused by epithelium removal can be estimated as follows: ACh X- MCh > BCh 2 Cch (Hoiroyde, 19% iriate et al., l9W, Small et a~., 1990). In spite of the pharmacological evidence, Small et al. (1990) recently reported that the guinea-pig tracheal epithelium is devoid of cholinesterase activity. It is possible that by mechanically removing the epithelium we inhibited chohnesterase activity in the vicinity of the smooth muscle. This seems unlikely because the cholinergic response to EFS, which is very sensitive to ace~lcholinesterase inhibition (D‘Agostino et al., 199Of, was unaffected by mechanical damage to the enithr_I--- hum. Although our findings are not consistent with the histologically demonstrated absence of both acetyicholinesterase and pseudochoiinesterase in the tracheal epithelium as described by Small et at. 119901,we postulate the possible involvement of cholinesterase activity in the epitheiial tissue on the basis of our results and of pha~acoIogica1 evidence (Taylor, 1990). This postulate will be tested by further experiments with butyrylchoiine, which is more likely to be hydrolyzed by pseudocholinesterase (butyryicholinesterasel than is ACh. Removal of the epithelium ~tentiated the histamine-induced contraction 4- to 7-fold. In epithcliumdenuded preparations, however, physostigmine (30 nMl did not attenuate the histamine-induced potentiation. It was recently reported that the epithelium decreased the release of histamine by allergen, probably due to metabolism of his&mine in guinea-pig trachea (Grundstrijm et aI., 1990). Thus, histamine-induced ~tentiation in epithelium-denuded preparations is likely to be due to either metabolism (by histaminase) or to a diffusion barrier effect of epithelium and/or EpDRF. The EpDRF, however, has not been biochemically identified as yet (Fernandes and Goldie, 19901. The results suggest strongly that the potentiation of the effect of exogenous contractile agonists by epitheiium removal may be due to a decrease in their metabolism In conclusion, the evidence indicates that the tracheal epitheiium inhibits ACh-induced contractions by serving as a source of acetylchoiinestcrase.
The authors wish lo thank Bradley J, Undcm, Ph.D.. The J&as Hopkins Un~ersity Asthma and Allergic Center. Baltimore, U.S.A. for critically rrvic@ing the ~nu~crip~.
llll.
Y.
and
K.
Tajima.
IqXi.
Action
pr~~~t~~~l~~n~i~~~ on ncuro-effector J. Physictl. fl.ondnnf Kocllz.
G.B..
lo&d
in~~~rn~ti~~~in
Basis of Thcrapcutics, (Macmillan.
itgent