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Effects of Na+ transport inhibitors on guinea-pig tracheal responses to spasmogens
European Jmtrnal of Pharmacology,
22 1 ( 1992) 43-50
43
0 1992 Elsevier Science Publishers B.V. All rights reserved 0014-2999/92/$05.00
EJP 526bX
Effects of Na+ transport inhibitors on guinea-pig tracheal responses to spasmogens Julio Cortijo, Departammt
Martin Gonzalez,
de Farmacologia.
Jo& L. Ortiz and Esteban
Faculto: dc Medicina,
Uniwrsilat
de Valhcia.
J. Morcillo Valkia.
Spain
Received 6 February 1992, revised MS received 15 May 1992, accepted 7 July 1992
The effects of ouabain, amiloride, Kt-free solution and low Nat (25 mM) solution on the responses to CaCI, (in Cal+-free, K+-depolarizing solution), KCI, acctylcholine, histamine and 5-hydroxytryptamine were studied in guinea-pig isolated trachea. Ouabain (10 FM) did not alter the contractile responses to CaCI,, KCI and acetylcholine but depressed those to histamine and S-hydroxytryptaminc produced in normal Ca’+ (2 .5 mM) and Ca*+-free (EGTA 0.1 mM) media. Amiloridc (0.1 mM), K+-free solution, and low Na+ solution depressed responses to acetylcholinc, histamine and 5-hydroxytryptamine produced in normal Ca’+ and Ca”+ -free media. Ouabain and amiloride had no effect on responses of skinned strips to Ca”. The mechanism of the inhibitory effects of these interventions is uncertain but the ‘findings suggest that the availability of Na+ influences the ahway smooth muscle responses to spasmogcns. Trachea (guinea-pig); Acetylcholine; Histamine; S-HTtS-hydroxytryptamine,
1. Introduction Force generation by smooth muscle ceils of the respiratory tract is regulated by the concentration of cytosolic free Ca2+ (Rodger and Small, 1991). Evidence exists that variations in intracellular or external Na+ concentration affect smooth muscle contractility by altering the levels of cytoplasmic free Ca2+ Wan Breemen et al., 1979). The existence of an active electrogenic Na+ pump has been demonstrated in airway smooth muscle (Souhrada et al., 1981). Inhibition of the Na+ pump by various pharmacologic interventions results in airway contraction in vivo (Marco et al., 1968) and in vitro (Souhrada and Souhrada. 1982; Black et al., 1984; Chideckel et al., 1987; Knox et al., 1990; Ortiz et al., 1991) in experimental animals and humans. However, inhaled ouabain, a Na+/K+ ATPase inhibitor (Fleming, 1980). failed to produce consistent bronchoconstriction in man (Agrawal and Hyatt, 1986; Knox et al., 1988). Reduction of external Na+ elicits spasm of airway muscle from a variety of mammalian species, including
Correspondence to: J. Cortijo, Departamcnt de Farmacologia, Facultat de Medicina. Avenida Blasco lbaiiez 15, 46010 Vakncia, Spain.
serotonin); Amiloride; Ouabain; K+-free solution
humans (Bullock et al., 1981; Kawanishi et al., 1984; Chideckel et al., 1987). This finding has been ascribed to a Na+/Ca’+ exchange mechanism in airway smooth muscle cells (Slaughter et al., 1987). Sarcolemmal Na+ transport systems may be important in determining airway muscle reactivity. A high dietary salt intake increases bronchial reactivity (Burney, 1987; Javaid et al., 1988; Burney et al., 1989). Inhaled ouabain increases histamine reactivity in conscious guinea-pigs (Agrawal and Hyatt, 1986) but not in humans (Knox et al., 1988). In vitro studies have shown that ouabain affects diversely the responsiveness and sensitivity of isolated airway preparations to a variety of spasmogens (Black et al., 1984; Raeburn and Fedan, 1989; Knox et al., 1990). The influence of other interventions which inhibit Na’ transport mechanisms in airway responses to spasmogens has not been reported upon. The aim of the present study was to investigate the effects of pharmacological interventions which inhibit the electrogenic Nat pump (ouabain, K+-free solution; Fleming, 1980; Souhrada et al., 1981) and the NaC/ Ca2+ exchange process (amiloride, low Na+ medium; Reuter et al., 1973; Benos, 1981; Bova et al., 1988) on guinea-pig tracheal responses in vitro to CaCI, (hi a Ca’+-free. K+-depolarizing solution), KCI. acetylcho!ine, histamine and 5hydroxytryptamine.
tion was 4S min b,i.)re
2. MtiltcrbIs and methods
and during the generation
the second concentration-effect curve. Two consecutive concentration-effect
Guinea-pigs of cithcr scs. weighing 4W-500 g were killed by stunning and b&ding. The tracheas WC~C escisai. cleaned of adhering tissues and cut into rings of ~~ppr~~~irn~t~l~ s-mm width. The rings wcrc then opened through the cartilage and the resulting tracheal strips were suspended in tissue baths containing a
curves
for
histamine (IO nM-I mM) wcrc obtained in a separate group of experiments. Second curves in test tissues were made in the absence (time-matched controls), or presence of phorbol 12,lIdiacetate (PDA; 5 p Ml, ouabain
(IO
FM).
amiloride
(0.1
mM),
ouabain
(10
PM) plus PDA (S MM). or amiloride (0.1 mM) plus PDA (5 ELM). Drugs were present in the bath f
ph~si~~logical salt soIutir?n (PSS, c~~rnpos~tion in mM:
min before and during the second concentration-effect
NaCl 115.4, KC1 4.7. NaHCO3 25.0. CaCI, 2.5 KH,PO, 1.2. MgSO, 0.6. glucose 11.1) maintained at F’C and continuously bubbled with 55 CO, in oxygen
curves.
tpH = 7.l). Great cart was taken during pr~par~~ti~?nof the tracheal strips nc?t ti? injure the cpithclium. An isometric recording of tension changes was obtained with force transducers (Grass FTW)
conncctcd through
of
(I mM),
Two succcssivc challcngcs with acetylcholinc bistaminc
(1 mM)
or S-hydrt?xytryptaminc
(0.1
mM)
~~n~pl~fi~~ tt? a ~?Iygraph (Grass 70). The strips wcrt’ gently stretched up to a l-g optimal resting tension (Ortiz ct al.. IWI) and a Wmin cquili-
were carried out. The first challenge was carried out in
hration period was allowed. with c’:angcs of the PSS at 15min intervals, bef(?rc any ph~~rm~?c~?l~?gici~l intententinn.
free, EGTA (0.1 mM) sitlulion containing or lacking ouahain (IO FM) or amiloride (0.1 mM). In addition,
PSS (Ca’ + 2.5 mM). Second challenges were obtained aftor Wmin incubation and in the prcscncc of a Cal?‘-
second challcngcs wcrc carried out in a Ca’+-free, K+-free, EGTA (0.1 mM)-containing solution or in a Ca’*-free. low Na’ (25 mM), EGTA fO.1 mM)-containing st?lution. The period of exposure to the modi-
Two
succcssivc
cumulative
conccntrrltk?n-~ffcct
curvt’s were r&t&cd for C&l, (0.1-I(10 mM). KC1 (~l.l-l(~) mM). ~~cctylch~?iincf I nM-IO mM1. histamine
ficd solutions was 45 mirl hcforc and during the gcncration of the second challangc. The spasmogenic effect in the second challenge was cxprcsscd as a proportion (5 1 of that in the first challenge.
4IO nM-I
mM) or Z-hydroxytryptaminc i 1 nM-0.1 mM). The expcriita
tion Ciarrii ct al.. 1989) in which K’
was suhstitutcd for Na * and Tris 5 mM rcplaccd NaHCO? as the buffer. The csporimcnts with the other spasmogcns tvcrc car&d out in PSS.
Segments of trachea were skinned of their plasmalcmmal mcmbrancs as previously reported (Ctrrtijo et al., IWJ).
Tissue scgmcnls were incubated
(4 h at
After an initial concentration-cffcct curve (first curve) for one of these spasmogcns had hccn obtained, the tissues were allocated randomly in cqua! numhcrs
3°C) in a 1% (v/v) Triton X-100 solution which contained (mM): KCI SO, sucrose 150, EGTA 5, imidazolc
tu test or to control groups and a sccttnd c~?nccntr~~tion-effect curve was ohtaincd.
IS min without
Second CUIVL’S in the test tissues wcrc obtained in the prcscncc of ouabain ( 10 g M) or amiloridc (0. I mMk The inhibit~~r was prcscnt in test tissues for 30
solution of (mM): EGTA 4. M&l, 10. ATP 7.5. NaN, I, imidazolc 20 and dithiocrythritol 0.5 (p1-I = 6.7), with 50% glycerol at - 211°C for up to 10 days. Under an
min heforc and during the second concentration-cffcct CUNC. Second CUNCS for acctylcholinc. histamine or S-hydm.xytryptaminc were also obtained in a K+-free
imposed tension of 1 g. segments of skinned trachea wcrc set up at 20°C f(?r isometric recording of tension
solution or a low-Na + 125 mMI solution. In addition, second curves for KC1 were generated in a low Na + solution. The K+-fret solution was prepared by rcmoving KCI from the PSS and replacing KH,PO, *,vith equim~~lar NaH,PO,. The h?w Na’ (25 mM) solution was prepared by isoosmotic rcplaccmcnt of NaCl by sucrc?sc. The period of cxposurc to the modified solu-
20 and dithiocrythritol
0.5 (pH = 7.4). After
rinsing for
in a ~~luti~~n of the same com~?siti~?n hut Triton X-100, the tissues wcrc stored in a
changes in 5 ml of relaxing solution containing
(mM):
EGTA 4, MgCI, 111,ATP 7.5, KH,PO, 6, NaN, 1 and imidazolc 20, adjusted to pH 6.7 with KOH. The relaxing solution did not contain added calmodulin. All tissues wcrc allowed to equilibrate in this medium for 20 min hcli-rrc c~~mmcncing the cumulative conccntration-effect curves for Ca’*. The amount of CaCI, to bc added to the relaxing
solution
to achieve
the re-
quired bath concentration
of free Ca’+
was calculated
as previously outlined (Cortijo et at., 1% ;i. Two successive concentration-effect curves for Ca*+ (0.2-!!!
pM) were obtained. In test tissues, ouabain (IO PM/~) or amiloride
tions are expressed as final bath concentrations of the active species. PDA was dissolved initially in dimethyl sulphoxide (Sigma), then diluted in distilled water and stored at -20°C.
(0.1 mM) was present for 30 min before and
throughout the second Ca*+ curve. Contn>l tissues were treated similarly except that they were not cx-
3.
Results
posed to the inhibitors. At the end of each experiment the preparation
was challenged with acetylcholinc
(100
3. I. Effects of pl~artt~acological ktert wtiotts ott baseline
PM). Ouabain (IO PM) produced an initial contraction with a peak at about 15 min followed by relaxation
2.5. Analysis of rcsult.~ Responses to spasmogens were generated from baseline. When a particular pharmacological intervention resulted
in a change of the resting tension which
was not fully dissipated
at the end of the incubation
period, mechanical readjustement was carried out to insure a similar value of the resting tension before challenging the tissue or obtaining a concentraiion-cffeet curve. The procedure to restore tone to the original lcvcl was necessary only for the experiments with amiloride. Preliminary results showed that the concentration-response curves obtained on addition of the spasmogen to tracheal strips contracted by amiloride did not significantly differ from those obtained after mechanical rcadjustmcnt to hasclinc (data not shown). The maximal cffcct (E,;,,) of a spasmogcn was expressed as mg of force developed per mg tissue (including the cartilage) dry weight (24 h at 64°C). The 50% cffectivc concentration (EC,,,) was calculated graphically from a plot of log concentration vs. % of E,,,;,, produced by each spasmogcn in individual expcrimcnts and was then transformed into -log,,, values for statistical purposes. The data arc prcscntcd as means + S.E.M. of n ckpcrimcnts. Statistical analysis of the results was pcrformed by analysis of variance (ANOVA) Duncan’s tests. Diffcrcnccs when P < 0.05.
followed by
wcrc considcrcd significant
2.6. Drug.~ ard soluliotu Acctylcholinc chloride, histamine hydrochloride, ouabain octahydratc and phorbo! 12,13-diacctatc wcrc purchased from Sigma Chemical Co. (St. Louis, MO, USA). Amiloride was from Merck, Sharp & Dohme (Madrid, Spain) and vcrapamil hydrochloride from Bioscdra-Knoll (Madrid, Spain). Other chemicals used were of analytical grade (E. Merck, Darmstadt, FRG; Panreac, Barcelona, Spain). The substances were dis-
below the resting tone then return to baseline in 25-30 min. Change of the PSS to a Caz+-free, EGTA (0.1 mM)-containing solution produced a small contraction with a peak in about 1-S min, followed by marked relaxation reaching a maximum after IO-IS min, with return to baseline by 20-30 min. Amiloride (0.1 mM) produced a contraction which reached its maximum in about IO min and remained above the resting tension after 30 min. Exposure to a K+ fret solution produced a transient (3-4 min) and small contraction followed by relaxation below the resting tone (maximum in IO-20 min) with return to baseline occurring by 30-40 min. Exposure to a low-Na+ (25 mM) solution resulted in contraction (peak in about 4-10 min) followed by relaxation below the resting tone with return to baseline occurring by 35-40
min.
_?.2. ~~&Ys of Na + tratrsporf itthibitot3 tmpot m’s IO spasttioget is Control cxpcrimcnts
ott trrrcheal
showed that conccntration-cf-
feet curves for CaCI, (in depolarizing tion) .I’ hisraminc ~~~Ly1~.rlUIlIIC, __-..,, KC!, ,,.-r..l..l-
CaZ+-fret soluand 5-hydroxy-
tryptaminc (first curves) did not change significantly when these spasmogens were retcstcd following 30-min incubation in the appropriate solution (second curves; see Methods). Ouabain did not alter the concentration-effect curves for CaCl,, KCI and acetylcholinc but produced a downward displacement of the curves for histamine and 5hydroxytryptaminc (fig. 1). The maximal cffccts of histamine and 5hydroxytryptaminc wcrc depressed in the prcscncc of ouabain whcrcas their EC,,, values were not significantly altered (table 1). Amiloridc did not affect concentration-cffcct CUIVCS for CaCI, and KCI while those for acctylcholinc. histamine and 5hydroxytryptamine wcrc displaced down-
solved in buffer solution just bcforc use. Vehicle controls (drug solvent only) were run in parallel; no signifi-
wards (fig. I). Amiloridc dccrcascd E,,;,, of accty!choline, histamine and S-hydroxytryptaminc but did not change the sensitivity of the preparations to the
cant vehicle cffccts were obscrvcd. With the cxccption of KC1 (whcrc the stated concentration was that in
spasmogcns tcstcd (table I). Concentration-effect curves for acctylcholinc.
cxccss of KCI from the buffer solution) drug conccntra-
taminc or 5-hydroxytryptaminc
ohtaincd
his-
in a K’-free
p
Q
.
r-v--f-c---
r-----?
1 98765_133
.l3~14J3
CaCi=
EC?
wh
-
--7--c
6 7 $15
I J
HA
I)
l
*’
e)-1’
A*
/
,”
A
.m.
.7---w
-_T-T-.
gH?ri’t
H 7 6 4 r,
,
-
t:t
z
T
6 /--
0’
lc
A_A l--R-“‘-,
R 7 6
./’*IA a--o*
r-1
5
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--
987654
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nn
IC’h
S-NT
,,
I
5-HT
Fig. I. The efftvt of au3bain (upl~r r0w ttf curves1 and smibride (b%Wr rOwI on tlic cl~ncr‘ntrr~~ion-eff~cl cuwcs for ChCI, (in H 13~ ‘-free. K+-depolarizing slUinnl_ KCI. acetylcholinr ( AChl. histamine (HAI and 5-hydrGrqtryptamine (%tlT) in the guinea-pig is&ted iruchea Two succesive mnccntr;ltir~n-effect CUNCS wwc &sincd. Initial curves (contmlsb are not shawn. Second cu~cs wcrc &tined in the skence (time matched r-~ntrrtls; 0) or prcsencc of auabain (I(1 PM: i!) CIT ;tmiktridc (0.1 mM: A 3. The results arc prescntcd as percentages d maGn;il responses o~hwd in the initial curves and arc means of six to eight experiments: S.E.M. shown h?:vertical bars. The abscissae indicate the concentration l~f the sp;lsmogcnsa - lag m&r (Mb crmcentration.
Fig. 2. The cFfect of K ‘-free solution k~pprt KIW of curves) and low Na ’ (25 mM) solution (lower rowf on the cclncentration-effect curves frlr ilcctylchc~line (ACh). histamine UlA) and S-hydroxytryptamine (S-11Tl in the guinea-pig isolated trachtm Two succesive cc~nccntration-rtffecr cuws were made. Initial curves (controls) are WI shown. Second CWKS were constructed in 6. absence (time mat&cd a>ntrols: *I or presence of K *-free solutia~~4.0) or It~w Na + s&&cm fn t. The results are presented as percentages of maximal responses ohtaincd in Be initial CUIWS ZIud are mean> of six to eight experiments: S.E.M. shown hy vertical bars. The abscissae indiqte 1he concentrations af the spasmogcns as -log molar (M) concentralion.
medium or a low Na” QS mM1 s&tion
the response to histamine II mM1 while it tended to be cnhanccd, failed to reach significance (fig. 3, Icft). E,;,, values of histamine were 373 L- 37 mg/mg (n = 6) in time-matched controls, 230 I!I 35 mg/mg in ouabaintreated tissues (P < &OS vs. control; n = li), and 270 -I33 mgfmg in tiua’oain pius PDA-treated tissues (P < &OS vs. control; n = 6). The changes in sensitivity were not significant. PDA displaced upward the conccntration-effect curves for histamine in the presence of
were shifted rightward and downward compared tu those obtained with time-matched control tissues (fig. 2, table I). The concentration-effect curve for KCi was not altered when gcncrttted in 3 low-Nrt ’ solution (tahlc 11. Pr~tr~~trngnc with PDA 15 JLM) did not affect the concentration-effect curve for histamine. The presetl~e of PDA did nut change the effect of ouabain an the concentration-effect curve for histamine although
TABLE
1
The effect crf exposure tn truahain (IO PM). amifctride (().I mM), ;1 K ‘-free s&lion. KCB. acctyicholine (.ArJhl, histamine (HA) and 5-hydroxytryptaminc (5HTI.
nr H low-Na ’ (75 mMl solutbn on the response ttr car) :-,
Responses to C‘aClz wcrlc obtained in a Gil”-free. dcpolarizinp (K ’ 55 mM) solution ilnd lh~~c to KU, acetylcholinc, histamine and C-hydroxytryptammr in PSS (Ca’ ’ 2.5 mM). Two successivecclncentr;rticln-effect curves were gcncra(Ld. First curved (cnntrols) arc not shown. Se~onrl curves were ohl;tined in the ahscncc (time-marched controls) or ptescnce of the intervrntions mentioned.
.. -____
Jw
CXI 2 E ma ”
- IW EC,,,
kc-matched control 177* 23 2.25+0161 Oushain (10 (1Ml 23.3* 2s I .Yh 5 (WI Time-matched control l79+ I:! 2. IS + 0.02 Amilnridc ((1.1 mM) 244+ ‘2 ~.Ihi().(12 Timc.m~tchcd cantml K’-free solution NDh Time-matchrd control Inw Na’ (3 mM) stWian ND
I%,.,,
- kg EC-q, E”,,,,
213+71) 2.lI(+(r.W1 ?I() 2 IS 2.33 f (1.08 250 + ‘5 2.03 f (I.09 303+ 26 l.92* 0.0s ND 149+22
HA
AC-h
1.9S_)(l.(l3
IIf>+ 1’) 2.10+0.07
S-NT
I%,,,,
- I(% ECli,,
I%,,
331 i46 4.x11* 0. I2 3W f 20 4.48 * 0. If, dhll f 23 4.sx f 0.22 223 +2x C .5.10~0.16 346 + 27 4.60~0.l2 234+2x’ 4.t13to.llc 270+24 4.9.3$(~.15
303 f 2s 207 * 24 p 324 f 24 20112 16 C 293&4I 129+12’ X0*31
&Yl *().I(> 4.hf>* O.O(l 3.98 y 0.07 s.o~c(t.07 XXI f 0. IS 4.32t+ft.06C 5. I3 -t_ 11.06
zlh*fI
154& 11’
102+20’
4.32f(WHC
- IW EC,,,
4.1(l~(l.l3’
- IW EC’s,,
h.40 * 0. IO I91 I 17 E S.hl & 0.20 309 * 23 6.28 + (12 1 2(12”k31 F &IX f(l.12
3lXrt34 ss*ll Xf I7
h.2(f+O.l7 5.46+(f.l5
Xhf21)
4.7()f().l2~
h.30 f 0. I (Y
a Maximal effect in mg force per ms of tissue dry weight. ” Not determined. The data WV means + S.E.M, trf six IO eight experiments fclr control and test tissues. P < il.05 cnmp;trcd ta cnntrnl.
47
* Central . . .
? 8
1
I
7
6
5
4
3
676543
Hiztamme
Fig. 3. The effects of phorbol 12,13-diacetate (PDA) and ouabain (left panel) or amiloride (right panel) on the concentralion-effect curves for histamine in guinea-pig isolated trachea. Two succesive concentration-effect curves to histamin.: were generated. Initial curves (control) are not shown. Second curves were made in the absence (control) or presence of ouabain (OUA), amiloride (AMI), PDA, ouabain plus PDA or amiloride plus PDA as indicated. The results are presented as percentages of maximal responses obtained in the
initial
curves and are
means
of six lo eight
(fig. 3, right). E,,, values of histamine were 324 + 24 mg/mg (n = 6) in time-matched controls, 175 + 28 mg/mg in amiloride-treated tissues (P < 0.05 VS. control; n = 6). and 201 + 27 mg/mg in amiloride plus PDA-treated tissues (P < 0.05 vs. amiloride-treated tissues; n = 6). When tracheal strips bathed in a Ca’+-free solution were challenged with acetylcholine (1 mM), histamine (I mM) or 5hydroxytryptamine (0.1 mM), only the contractions due to 5-hydroxytryptamine were depressed. Responses to acetylcholine, histamine and 5hydroxytryptamine in a Ca *+-free medium were inhibited further by incubation with ouabain (10 PM), amiloride (0.1 mM), a K+-free medium or a low Na+ solution. Ouabain failed to depress the contraction produced by acetylcholine in Ca”+-free solution (fig. 4). amiloride
PDA 5 /LM AMI 0 1 mM AMI + PDA
3.3. Effects of ouabain and amiloride on skinned tracheal preparatiom
experiments;
S.E.M. shown by vertical bars. The abscissae indicate the concentrations of the spasmogensas -log molar (MI concentration.
Segments of skinned trachea contracted in a concentration-related manner in response to Ca*+. The second concentration-effect curve for Caz+ obtained in the same preparation was slightly displaced to the left ( -log EC5,, for first curve was 6.31 f 0.03 vs. 6.44 + 0.04 for the second curve; n = 5; P < 0.05) with a small depression of its maximum (I 1.2 _t 5.6%) compared to the initial concentration-effect curve. Guabain (IO PM) and amiloride (0.1 mM) had _r~ effect on the sensitivity and maximum responses of skinned strips to Ca*+ (data not shown). The acetylcholine (100 PM) challenge terminating each experiment produced no tension increase.
4. Discussion 2
120 -
5-hydroxytryptamme
00
40
0 Contrul
AMI OUA 10 1rM ” I mM
K’frrr
Low N.,-
Fig. 4. The influence of ouabain (OLJA), amiloride (AMI). K’-free solution and low Na* (25 mM) solution on the contractile responses of the guinea-pig trachea to acetylcholine (I mM), histamine (I mM) or S-hydroxytryptamine (0.1 mM) in Ca*‘-free medium. Two succcssive challenges were generated. First challenges in normal CaZ’ (2.5 mM) PSS are not shown. Second challenges were in a &‘-free (EGTA 0.1 mM) solution in the absence (control) or presence of additions. Responses are presented as percentages of Ihat in Ihc first challenge. Column heights indicate the mean of six to eight experiments and vertical lines indicatethe S.E.M. * P < 0.05 compared to first challenge in PSS (Ca*’ 2.5 mM). ’ P < 0.05 compared to second challenge in a &‘-free medium.
The purpose of the present study was to determine whether interventions that affect membrane Na’ transport alter airway responses to spasmogens in vitro. Ouabain or external K+ removal, which inhibit Na+/K+-ATPase (Fleming, 1980), amiloride, which inhibits Na’ entry, Na’/H+ exchange and Na+/Ca’+ exchange (Reuter et al., 1973; Bcnos, 1981; Bova et al., l988), and reduction in trans-sarcolemmal Na+ gradient by exposure to a Nat-depleted medium were used. All these interventions, except amiloride produced a transient contraction followed by relaxation then return to baseline. Amiloride caused a sustained contraction. The mechanisms underlying these effects could involve, depending on the agent, Na+ loading, intracellular Ca*+ accumulation and other changes which have been reported and reviewed in the literature (Souhrada et al., 1981; Kawanishi ct ab, 1984; Knox et al., 1990). The spasmogens were selected to produce tracheal contraction predominantly due to either cxtracellular
us
qbnses
(3~ intracellular
to C&‘l.‘
~~~~ 41~ Ii;ct
etlap
Gil-’ ’
dCil!X.
‘I‘IIUS. rc-
(in Ca- +-free. K +-depolarizing
;trc the conscqucncc of cxtraccIhkIr
thmuph
voltage-dependent
Cal’
S~hl-
CO’ ’
channels
choline, histamine and S-hydroxytryptamine were generated in the absence of extraccllular Ca’+, ouabain did not affect acetylcholine-induced effective
to depressing
spasm but was as
responses to histamine
hydroxytryptamine
recuptors generate inositol I.6triphosphate which is respc>nsible for intracellular Ca’ + mobilization. and diac~;lglycer~~l which activates protein kinase C
others.
(S&amn and Grunstein. lY8Y: Rodgcr and Small. IWI ). Membrane depolarization. increase of intraccl-
Itoh, IYX4). An effect of ouabain on Ca’ ’ rclcase from sarct,plasnlic reticulum has been shown for vascular
lular Ca2 - and activation c>f protein by spasmogens may result. through
kiniWZ
C
different
nisms. in alterations of the trans-membrane ent. Na ‘-K * ATPase activity, and the Na
produced mccha-
Na ’ gradil /Ca’ + and
as in PSS. These
and S-
cpucd by dc~~larization (Foster et al.. 1YX.t: Rodgcr. lvzi7). hn clmtrast, agonists (acetylcholine. histamine. 5-hydn~qqryptaminc) that activate specific membrane
results
suggest
that ouabain may interfere with the intracellular Ca” rclcasc triggered by certain spasmogens but not by This may bc cxpkined by the cxistcncc of different intracellular Ca’ ’ stores available for diffcrcnt spasmogens. as previously shown by others (Ito and
smooth muscle (Lamb et al.. 1088). Results from the experiments with skinned
trachea
rule out the possibility that ouabain interfered
with the
intracellular contractile mechanism. Therefore, the inhibitory effect of ouabain has to be ascribed to the role
Na ‘/H + exchange systems (Schramn and Grunstcin, IYSY). Therefore. interventions which inhibit mem-
of intracellular
hranc
movements (Van Breemen et al.. 1070: Blaustein,
MI
+
transport could affect tracheal rcsponscs to
spilsmOgCIlS.
Ouahain (0.1 PM) has hccn reported to increase sensitivity of intact guinea-pip trachea to methacholinc but it dccrcascd the sensitivity to KC.1 (Racburn and I-cdan. 1’159). Ouabain did not alter methacholinc- and KCI-induced rcsponscs in cpithclium-denuded guincapig trachea (Raehurn and Fedan, IYSY). This indicates that integrity of the cpithclium is important to the effects of ouabain. All expcrimcnts in the present study were carried out in intact guinea-pig trachea. When the effect of a concentration of ouabain (10 PM) sufficient to fully inhibit Na’.K’-ATPase in guinea-pig trachea (Souhradd ct al.. IYXI) was studied on the rcqnmses to a variety ot spasmogens, it was found that only contractions due to histamine and S-hydroxylryptamine
were depressed while those due to CaCI , depolarizing so!ution). KC1 and ace&
!in Ca-“-free
choline remained unaffected. In iso,htcd human bronchi.
Black et al. (IYX4)
re-
ported that pretreatment with a high concentration of ouabain (SO PM) markedly attenuated the response :o KCI. carbachol and histamine but Knox et al. (1900) found that ouabain (IO PM) did not a!ter methacholine-induced responses. These results are in contrast with those in vascular smooth muscle where inhibition of the Na ‘.K’ pump potentiates contractions clicitcd by a variety of activating agents (Van Breemen et al.. lY7Y)_ The mechanism underlying the inhibitory effect of ouabain on airway responses to spasmogens in vitro is not clear. Interference with Ca” entry through voltage-dependent channels is ruled out since CaCl,- and KCI-induced responses are not affected. Moreover, the effects of ouabain and those of Ca’+ entry channel blockers on tracheal responsiveness to spasmogens arc not equivalent (Foster et al., 19X4; Advenier et al., 1YW this study). When maximal responses to acetyl-
Na + as modulator
of intracellular
Ca” IYXY).
Removal of external K’ also inhibits Na’/K+ATPasc but its clectrophysiological effects arc different from those observed after ouahain !Souhrada
ct al.,
19x1), Exposure to a K +-free solution dcprcssed responses to acetylcholine. tryptamine
the and S-hydroxy-
histamine
in PSS and C$+-free
solution. We have not
found other reports in the literature on these effects of K ‘-free media. The mechanism of the inhibitory effect of reduced external K’ is uncertain but considerations similar to those for ouabain are probably valid in this
rcspcct . Amiloride
depressed the responses to acetylcholine.
histamine
and S-hydroxytryptamine
Ca”-free
solution
in both
PSS and
(this study). These findings
are in
agreement with those of Knox et al. (IYYO) showing that amiloride (0.1-l mM) inhibited conlractions of bovine trachea produced by carhachol and histamine. Souhrada et al. (lYX8) reported that amiloride (IO or.M) prevent& contractile and electrical responses to spccific antigen challenge in isolated guinea-pig trachea. Unfortunately amiloride is not a selective pharmacological tool. Thus, amiloridc inhibits a Na + entry channel (Berms lY81). the Nat/H+ antiport (Benos, 1981) and Na’/Ca’+ exchange (Debetto et al. 1987) and voltage-dependent Ca’+ channels (Klcyman and Cragee. IY%H). The lack of effect of amiloride (0.1 mM) on CaCl,- and KCI-induced spasm rules out a mechanism rclatcd to inlcrfcrcncc of Ca’+ entry through voltagcdependent Ca’+ channels in this preparation. Amiloride may also inhibit protein kinase C activity (Besterman et al., lYt(S). PDA, an activator of protein kinase C (Nishizuka, lY84), partly re*lersed the inhibition produced
by amiloridc
(0.1
mM)
on histamine-induced
contraction (this study). This indicates that part of the effects of amiloride could be related to inhibition of protein with
kinase C. The a
greater
use of analogues
selectivity
for
the
of amiloride
Na ‘/H
+
and
-lY
Na +/Ca’+ exchangers Wigne ct al., 1983; Kaczorowski ct al., 1989) may help to prccisc the importancc of these systems in agonist-induced contraction of airways smooth muscle. Exposure to a low-Na + solution also depressed rcsponses to acetylcholine, histamine and S-hydroxytryptamine in PSS and Ca’+-free medium but KCI-in-
to dctcrminc
precisely
which
ccrrtijo. J.. J.S. Dixon.
Drhetto.
mcchanismb)
solution was simi-
mogens. In conclusion, interventions that affect membrane Nat transport dcprcsscd contractile rcsponscs to physiological substances of some importance in asthma.
hy emiloride. Fleming.
C’.I.C’.Y.T.
(Ministerio
in part
de Industria
hy Grunt
y Energia)
FARYII-6X0
Foster and R.C. Small.
from
Spain. The authors
thank for the v;rluahlC technical assistance of Mr. P. Santamitrin.
X-ItHI
from guinea-pig
F. Carpcnedo
IYXO. The
rlectrogrnic
Pharmacol.
20, 120.
Toxical. RI‘.
Sm;rll and A.H.
action of potassium chloride macol. XI). 5.53. Foster, R.W.. B.I. Okpalugo
lYX7. Inhihi-
in smooth
significance.
Weston.
and R.I’.
Chcalis. Br. J. Pharmacol.
Ann.
Rev.
IYX3. The spasmogenic
Small.
Br. J. Phar-
10X4. Antagonism
in guinea-pig
of
isolated tra-
Xl. 490.
10X4. The roles of stored calcium in contractions
trachC;ll smooth muscle produced hy elCctriC;ll stimulation.
ncetylcholinc
and high K
‘,
Br. J. PharmaCol. X.3, 6h7.
Javaid. A.. M.J. C’ushley and M.F. on bronchial 4%.
reactivity
G.J..
M.L.
Bone. IYXX. Effect of dietary salt
to histamine Garcia.
Noble and II. RKUlKr
(Oxford
University
M.,
K.
and
Baha
T.R. and E.J. Cragor,
19X4.
of Na’
inhaled ouahain
Effects response
and J.R. Britttrn,
on hrolrchial
in the
Jap. J. Physiol. 34. 127.
IYXX. Amiloride
Tattersficld
D.
p. (6
and its analogurs as
tools in the study of ion transptrrt. J. Memhr. Knox. A.J.. A.E.
KX-
Allen.
Press. Oxford)
cm thC mechanical
smooth mUSClK.
puinca-pig tracheal
eds. T.J.A.
and 1’. Tomita,
readmission
IYXY.
to study sodium-calcium
Exchange.
rcmovul
297.
F. King and R.S. Slaughter.
and use of inhihitors Calcium
Kawanishi.
Br. J. Meti.
in asthma,
in: Sodium
Klrymun.
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This
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was responsible for the effects observed in this study. The pattern of changes in the responses to spasmogens produced by cxposurc to a low-Na+
on lension
pl;~smalcmm;d mcmhranc
The effects of reducing extracellular Nat on responses to spasmogens has not been studied previously and it is difficult
E.W.. J.L. Froxt, P. Mike and J.S. Fedan.
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of somC varlahles
in PSS was not affected (this study).
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K.P.
A. Floch and A. ReniCr,
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IYX4.
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from
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;Ind G. IbeuslCr.
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R.S.
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Rcspir. Physiol. 57. 260.
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effects of digitalis in the anarstllesized
E.J. Cragoe and P. Cuatre-
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Bonus. D.J.. IYXI. Amiloridc: Brstrrman,
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Souhrada. M., M.H. Souhrada and J.F. Souhrirda. 19X8, The inhibition of sodium influx attenuates airway response to a specific antigen challenge, Br. J. Pharrnacol. 93. X84. Van Breemen, C.. P. Aaronson and R. Loutzenhiser. 1979. Sodiumcalcium interactions in mammnlinn smooth muscle. Pharmacol. Rev. 30, lh7. Vigne, P.. C. Frelin. E.J. Cragoe and M. Ladzunski, 1983, Structureactivity relationships of amiloride and certain of its analogues in relation to the blockade of the Nat/H ’ exchange system, Mo! Pharmiicol. 2S. 131.
22 1 ( 1992) 43-50
43
0 1992 Elsevier Science Publishers B.V. All rights reserved 0014-2999/92/$05.00
EJP 526bX
Effects of Na+ transport inhibitors on guinea-pig tracheal responses to spasmogens Julio Cortijo, Departammt
Martin Gonzalez,
de Farmacologia.
Jo& L. Ortiz and Esteban
Faculto: dc Medicina,
Uniwrsilat
de Valhcia.
J. Morcillo Valkia.
Spain
Received 6 February 1992, revised MS received 15 May 1992, accepted 7 July 1992
The effects of ouabain, amiloride, Kt-free solution and low Nat (25 mM) solution on the responses to CaCI, (in Cal+-free, K+-depolarizing solution), KCI, acctylcholine, histamine and 5-hydroxytryptamine were studied in guinea-pig isolated trachea. Ouabain (10 FM) did not alter the contractile responses to CaCI,, KCI and acetylcholine but depressed those to histamine and S-hydroxytryptaminc produced in normal Ca’+ (2 .5 mM) and Ca*+-free (EGTA 0.1 mM) media. Amiloridc (0.1 mM), K+-free solution, and low Na+ solution depressed responses to acetylcholinc, histamine and 5-hydroxytryptamine produced in normal Ca’+ and Ca”+ -free media. Ouabain and amiloride had no effect on responses of skinned strips to Ca”. The mechanism of the inhibitory effects of these interventions is uncertain but the ‘findings suggest that the availability of Na+ influences the ahway smooth muscle responses to spasmogcns. Trachea (guinea-pig); Acetylcholine; Histamine; S-HTtS-hydroxytryptamine,
1. Introduction Force generation by smooth muscle ceils of the respiratory tract is regulated by the concentration of cytosolic free Ca2+ (Rodger and Small, 1991). Evidence exists that variations in intracellular or external Na+ concentration affect smooth muscle contractility by altering the levels of cytoplasmic free Ca2+ Wan Breemen et al., 1979). The existence of an active electrogenic Na+ pump has been demonstrated in airway smooth muscle (Souhrada et al., 1981). Inhibition of the Na+ pump by various pharmacologic interventions results in airway contraction in vivo (Marco et al., 1968) and in vitro (Souhrada and Souhrada. 1982; Black et al., 1984; Chideckel et al., 1987; Knox et al., 1990; Ortiz et al., 1991) in experimental animals and humans. However, inhaled ouabain, a Na+/K+ ATPase inhibitor (Fleming, 1980). failed to produce consistent bronchoconstriction in man (Agrawal and Hyatt, 1986; Knox et al., 1988). Reduction of external Na+ elicits spasm of airway muscle from a variety of mammalian species, including
Correspondence to: J. Cortijo, Departamcnt de Farmacologia, Facultat de Medicina. Avenida Blasco lbaiiez 15, 46010 Vakncia, Spain.
serotonin); Amiloride; Ouabain; K+-free solution
humans (Bullock et al., 1981; Kawanishi et al., 1984; Chideckel et al., 1987). This finding has been ascribed to a Na+/Ca’+ exchange mechanism in airway smooth muscle cells (Slaughter et al., 1987). Sarcolemmal Na+ transport systems may be important in determining airway muscle reactivity. A high dietary salt intake increases bronchial reactivity (Burney, 1987; Javaid et al., 1988; Burney et al., 1989). Inhaled ouabain increases histamine reactivity in conscious guinea-pigs (Agrawal and Hyatt, 1986) but not in humans (Knox et al., 1988). In vitro studies have shown that ouabain affects diversely the responsiveness and sensitivity of isolated airway preparations to a variety of spasmogens (Black et al., 1984; Raeburn and Fedan, 1989; Knox et al., 1990). The influence of other interventions which inhibit Na’ transport mechanisms in airway responses to spasmogens has not been reported upon. The aim of the present study was to investigate the effects of pharmacological interventions which inhibit the electrogenic Nat pump (ouabain, K+-free solution; Fleming, 1980; Souhrada et al., 1981) and the NaC/ Ca2+ exchange process (amiloride, low Na+ medium; Reuter et al., 1973; Benos, 1981; Bova et al., 1988) on guinea-pig tracheal responses in vitro to CaCI, (hi a Ca’+-free. K+-depolarizing solution), KCI. acetylcho!ine, histamine and 5hydroxytryptamine.
tion was 4S min b,i.)re
2. MtiltcrbIs and methods
and during the generation
the second concentration-effect curve. Two consecutive concentration-effect
Guinea-pigs of cithcr scs. weighing 4W-500 g were killed by stunning and b&ding. The tracheas WC~C escisai. cleaned of adhering tissues and cut into rings of ~~ppr~~~irn~t~l~ s-mm width. The rings wcrc then opened through the cartilage and the resulting tracheal strips were suspended in tissue baths containing a
curves
for
histamine (IO nM-I mM) wcrc obtained in a separate group of experiments. Second curves in test tissues were made in the absence (time-matched controls), or presence of phorbol 12,lIdiacetate (PDA; 5 p Ml, ouabain
(IO
FM).
amiloride
(0.1
mM),
ouabain
(10
PM) plus PDA (S MM). or amiloride (0.1 mM) plus PDA (5 ELM). Drugs were present in the bath f
ph~si~~logical salt soIutir?n (PSS, c~~rnpos~tion in mM:
min before and during the second concentration-effect
NaCl 115.4, KC1 4.7. NaHCO3 25.0. CaCI, 2.5 KH,PO, 1.2. MgSO, 0.6. glucose 11.1) maintained at F’C and continuously bubbled with 55 CO, in oxygen
curves.
tpH = 7.l). Great cart was taken during pr~par~~ti~?nof the tracheal strips nc?t ti? injure the cpithclium. An isometric recording of tension changes was obtained with force transducers (Grass FTW)
conncctcd through
of
(I mM),
Two succcssivc challcngcs with acetylcholinc bistaminc
(1 mM)
or S-hydrt?xytryptaminc
(0.1
mM)
~~n~pl~fi~~ tt? a ~?Iygraph (Grass 70). The strips wcrt’ gently stretched up to a l-g optimal resting tension (Ortiz ct al.. IWI) and a Wmin cquili-
were carried out. The first challenge was carried out in
hration period was allowed. with c’:angcs of the PSS at 15min intervals, bef(?rc any ph~~rm~?c~?l~?gici~l intententinn.
free, EGTA (0.1 mM) sitlulion containing or lacking ouahain (IO FM) or amiloride (0.1 mM). In addition,
PSS (Ca’ + 2.5 mM). Second challenges were obtained aftor Wmin incubation and in the prcscncc of a Cal?‘-
second challcngcs wcrc carried out in a Ca’+-free, K+-free, EGTA (0.1 mM)-containing solution or in a Ca’*-free. low Na’ (25 mM), EGTA fO.1 mM)-containing st?lution. The period of exposure to the modi-
Two
succcssivc
cumulative
conccntrrltk?n-~ffcct
curvt’s were r&t&cd for C&l, (0.1-I(10 mM). KC1 (~l.l-l(~) mM). ~~cctylch~?iincf I nM-IO mM1. histamine
ficd solutions was 45 mirl hcforc and during the gcncration of the second challangc. The spasmogenic effect in the second challenge was cxprcsscd as a proportion (5 1 of that in the first challenge.
4IO nM-I
mM) or Z-hydroxytryptaminc i 1 nM-0.1 mM). The expcriita
tion Ciarrii ct al.. 1989) in which K’
was suhstitutcd for Na * and Tris 5 mM rcplaccd NaHCO? as the buffer. The csporimcnts with the other spasmogcns tvcrc car&d out in PSS.
Segments of trachea were skinned of their plasmalcmmal mcmbrancs as previously reported (Ctrrtijo et al., IWJ).
Tissue scgmcnls were incubated
(4 h at
After an initial concentration-cffcct curve (first curve) for one of these spasmogcns had hccn obtained, the tissues were allocated randomly in cqua! numhcrs
3°C) in a 1% (v/v) Triton X-100 solution which contained (mM): KCI SO, sucrose 150, EGTA 5, imidazolc
tu test or to control groups and a sccttnd c~?nccntr~~tion-effect curve was ohtaincd.
IS min without
Second CUIVL’S in the test tissues wcrc obtained in the prcscncc of ouabain ( 10 g M) or amiloridc (0. I mMk The inhibit~~r was prcscnt in test tissues for 30
solution of (mM): EGTA 4. M&l, 10. ATP 7.5. NaN, I, imidazolc 20 and dithiocrythritol 0.5 (p1-I = 6.7), with 50% glycerol at - 211°C for up to 10 days. Under an
min heforc and during the second concentration-cffcct CUNC. Second CUNCS for acctylcholinc. histamine or S-hydm.xytryptaminc were also obtained in a K+-free
imposed tension of 1 g. segments of skinned trachea wcrc set up at 20°C f(?r isometric recording of tension
solution or a low-Na + 125 mMI solution. In addition, second curves for KC1 were generated in a low Na + solution. The K+-fret solution was prepared by rcmoving KCI from the PSS and replacing KH,PO, *,vith equim~~lar NaH,PO,. The h?w Na’ (25 mM) solution was prepared by isoosmotic rcplaccmcnt of NaCl by sucrc?sc. The period of cxposurc to the modified solu-
20 and dithiocrythritol
0.5 (pH = 7.4). After
rinsing for
in a ~~luti~~n of the same com~?siti~?n hut Triton X-100, the tissues wcrc stored in a
changes in 5 ml of relaxing solution containing
(mM):
EGTA 4, MgCI, 111,ATP 7.5, KH,PO, 6, NaN, 1 and imidazolc 20, adjusted to pH 6.7 with KOH. The relaxing solution did not contain added calmodulin. All tissues wcrc allowed to equilibrate in this medium for 20 min hcli-rrc c~~mmcncing the cumulative conccntration-effect curves for Ca’*. The amount of CaCI, to bc added to the relaxing
solution
to achieve
the re-
quired bath concentration
of free Ca’+
was calculated
as previously outlined (Cortijo et at., 1% ;i. Two successive concentration-effect curves for Ca*+ (0.2-!!!
pM) were obtained. In test tissues, ouabain (IO PM/~) or amiloride
tions are expressed as final bath concentrations of the active species. PDA was dissolved initially in dimethyl sulphoxide (Sigma), then diluted in distilled water and stored at -20°C.
(0.1 mM) was present for 30 min before and
throughout the second Ca*+ curve. Contn>l tissues were treated similarly except that they were not cx-
3.
Results
posed to the inhibitors. At the end of each experiment the preparation
was challenged with acetylcholinc
(100
3. I. Effects of pl~artt~acological ktert wtiotts ott baseline
PM). Ouabain (IO PM) produced an initial contraction with a peak at about 15 min followed by relaxation
2.5. Analysis of rcsult.~ Responses to spasmogens were generated from baseline. When a particular pharmacological intervention resulted
in a change of the resting tension which
was not fully dissipated
at the end of the incubation
period, mechanical readjustement was carried out to insure a similar value of the resting tension before challenging the tissue or obtaining a concentraiion-cffeet curve. The procedure to restore tone to the original lcvcl was necessary only for the experiments with amiloride. Preliminary results showed that the concentration-response curves obtained on addition of the spasmogen to tracheal strips contracted by amiloride did not significantly differ from those obtained after mechanical rcadjustmcnt to hasclinc (data not shown). The maximal cffcct (E,;,,) of a spasmogcn was expressed as mg of force developed per mg tissue (including the cartilage) dry weight (24 h at 64°C). The 50% cffectivc concentration (EC,,,) was calculated graphically from a plot of log concentration vs. % of E,,,;,, produced by each spasmogcn in individual expcrimcnts and was then transformed into -log,,, values for statistical purposes. The data arc prcscntcd as means + S.E.M. of n ckpcrimcnts. Statistical analysis of the results was pcrformed by analysis of variance (ANOVA) Duncan’s tests. Diffcrcnccs when P < 0.05.
followed by
wcrc considcrcd significant
2.6. Drug.~ ard soluliotu Acctylcholinc chloride, histamine hydrochloride, ouabain octahydratc and phorbo! 12,13-diacctatc wcrc purchased from Sigma Chemical Co. (St. Louis, MO, USA). Amiloride was from Merck, Sharp & Dohme (Madrid, Spain) and vcrapamil hydrochloride from Bioscdra-Knoll (Madrid, Spain). Other chemicals used were of analytical grade (E. Merck, Darmstadt, FRG; Panreac, Barcelona, Spain). The substances were dis-
below the resting tone then return to baseline in 25-30 min. Change of the PSS to a Caz+-free, EGTA (0.1 mM)-containing solution produced a small contraction with a peak in about 1-S min, followed by marked relaxation reaching a maximum after IO-IS min, with return to baseline by 20-30 min. Amiloride (0.1 mM) produced a contraction which reached its maximum in about IO min and remained above the resting tension after 30 min. Exposure to a K+ fret solution produced a transient (3-4 min) and small contraction followed by relaxation below the resting tone (maximum in IO-20 min) with return to baseline occurring by 30-40 min. Exposure to a low-Na+ (25 mM) solution resulted in contraction (peak in about 4-10 min) followed by relaxation below the resting tone with return to baseline occurring by 35-40
min.
_?.2. ~~&Ys of Na + tratrsporf itthibitot3 tmpot m’s IO spasttioget is Control cxpcrimcnts
ott trrrcheal
showed that conccntration-cf-
feet curves for CaCI, (in depolarizing tion) .I’ hisraminc ~~~Ly1~.rlUIlIIC, __-..,, KC!, ,,.-r..l..l-
CaZ+-fret soluand 5-hydroxy-
tryptaminc (first curves) did not change significantly when these spasmogens were retcstcd following 30-min incubation in the appropriate solution (second curves; see Methods). Ouabain did not alter the concentration-effect curves for CaCl,, KCI and acetylcholinc but produced a downward displacement of the curves for histamine and 5hydroxytryptaminc (fig. 1). The maximal cffccts of histamine and 5hydroxytryptaminc wcrc depressed in the prcscncc of ouabain whcrcas their EC,,, values were not significantly altered (table 1). Amiloridc did not affect concentration-cffcct CUIVCS for CaCI, and KCI while those for acctylcholinc. histamine and 5hydroxytryptamine wcrc displaced down-
solved in buffer solution just bcforc use. Vehicle controls (drug solvent only) were run in parallel; no signifi-
wards (fig. I). Amiloridc dccrcascd E,,;,, of accty!choline, histamine and S-hydroxytryptaminc but did not change the sensitivity of the preparations to the
cant vehicle cffccts were obscrvcd. With the cxccption of KC1 (whcrc the stated concentration was that in
spasmogcns tcstcd (table I). Concentration-effect curves for acctylcholinc.
cxccss of KCI from the buffer solution) drug conccntra-
taminc or 5-hydroxytryptaminc
ohtaincd
his-
in a K’-free
p
Q
.
r-v--f-c---
r-----?
1 98765_133
.l3~14J3
CaCi=
EC?
wh
-
--7--c
6 7 $15
I J
HA
I)
l
*’
e)-1’
A*
/
,”
A
.m.
.7---w
-_T-T-.
gH?ri’t
H 7 6 4 r,
,
-
t:t
z
T
6 /--
0’
lc
A_A l--R-“‘-,
R 7 6
./’*IA a--o*
r-1
5
.I
--
987654
3
nn
IC’h
S-NT
,,
I
5-HT
Fig. I. The efftvt of au3bain (upl~r r0w ttf curves1 and smibride (b%Wr rOwI on tlic cl~ncr‘ntrr~~ion-eff~cl cuwcs for ChCI, (in H 13~ ‘-free. K+-depolarizing slUinnl_ KCI. acetylcholinr ( AChl. histamine (HAI and 5-hydrGrqtryptamine (%tlT) in the guinea-pig is&ted iruchea Two succesive mnccntr;ltir~n-effect CUNCS wwc &sincd. Initial curves (contmlsb are not shawn. Second cu~cs wcrc &tined in the skence (time matched r-~ntrrtls; 0) or prcsencc of auabain (I(1 PM: i!) CIT ;tmiktridc (0.1 mM: A 3. The results arc prescntcd as percentages d maGn;il responses o~hwd in the initial curves and arc means of six to eight experiments: S.E.M. shown h?:vertical bars. The abscissae indicate the concentration l~f the sp;lsmogcnsa - lag m&r (Mb crmcentration.
Fig. 2. The cFfect of K ‘-free solution k~pprt KIW of curves) and low Na ’ (25 mM) solution (lower rowf on the cclncentration-effect curves frlr ilcctylchc~line (ACh). histamine UlA) and S-hydroxytryptamine (S-11Tl in the guinea-pig isolated trachtm Two succesive cc~nccntration-rtffecr cuws were made. Initial curves (controls) are WI shown. Second CWKS were constructed in 6. absence (time mat&cd a>ntrols: *I or presence of K *-free solutia~~4.0) or It~w Na + s&&cm fn t. The results are presented as percentages of maximal responses ohtaincd in Be initial CUIWS ZIud are mean> of six to eight experiments: S.E.M. shown hy vertical bars. The abscissae indiqte 1he concentrations af the spasmogcns as -log molar (M) concentralion.
medium or a low Na” QS mM1 s&tion
the response to histamine II mM1 while it tended to be cnhanccd, failed to reach significance (fig. 3, Icft). E,;,, values of histamine were 373 L- 37 mg/mg (n = 6) in time-matched controls, 230 I!I 35 mg/mg in ouabaintreated tissues (P < &OS vs. control; n = li), and 270 -I33 mgfmg in tiua’oain pius PDA-treated tissues (P < &OS vs. control; n = 6). The changes in sensitivity were not significant. PDA displaced upward the conccntration-effect curves for histamine in the presence of
were shifted rightward and downward compared tu those obtained with time-matched control tissues (fig. 2, table I). The concentration-effect curve for KCi was not altered when gcncrttted in 3 low-Nrt ’ solution (tahlc 11. Pr~tr~~trngnc with PDA 15 JLM) did not affect the concentration-effect curve for histamine. The presetl~e of PDA did nut change the effect of ouabain an the concentration-effect curve for histamine although
TABLE
1
The effect crf exposure tn truahain (IO PM). amifctride (().I mM), ;1 K ‘-free s&lion. KCB. acctyicholine (.ArJhl, histamine (HA) and 5-hydroxytryptaminc (5HTI.
nr H low-Na ’ (75 mMl solutbn on the response ttr car) :-,
Responses to C‘aClz wcrlc obtained in a Gil”-free. dcpolarizinp (K ’ 55 mM) solution ilnd lh~~c to KU, acetylcholinc, histamine and C-hydroxytryptammr in PSS (Ca’ ’ 2.5 mM). Two successivecclncentr;rticln-effect curves were gcncra(Ld. First curved (cnntrols) arc not shown. Se~onrl curves were ohl;tined in the ahscncc (time-marched controls) or ptescnce of the intervrntions mentioned.
.. -____
Jw
CXI 2 E ma ”
- IW EC,,,
kc-matched control 177* 23 2.25+0161 Oushain (10 (1Ml 23.3* 2s I .Yh 5 (WI Time-matched control l79+ I:! 2. IS + 0.02 Amilnridc ((1.1 mM) 244+ ‘2 ~.Ihi().(12 Timc.m~tchcd cantml K’-free solution NDh Time-matchrd control Inw Na’ (3 mM) stWian ND
I%,.,,
- kg EC-q, E”,,,,
213+71) 2.lI(+(r.W1 ?I() 2 IS 2.33 f (1.08 250 + ‘5 2.03 f (I.09 303+ 26 l.92* 0.0s ND 149+22
HA
AC-h
1.9S_)(l.(l3
IIf>+ 1’) 2.10+0.07
S-NT
I%,,,,
- I(% ECli,,
I%,,
331 i46 4.x11* 0. I2 3W f 20 4.48 * 0. If, dhll f 23 4.sx f 0.22 223 +2x C .5.10~0.16 346 + 27 4.60~0.l2 234+2x’ 4.t13to.llc 270+24 4.9.3$(~.15
303 f 2s 207 * 24 p 324 f 24 20112 16 C 293&4I 129+12’ X0*31
&Yl *().I(> 4.hf>* O.O(l 3.98 y 0.07 s.o~c(t.07 XXI f 0. IS 4.32t+ft.06C 5. I3 -t_ 11.06
zlh*fI
154& 11’
102+20’
4.32f(WHC
- IW EC,,,
4.1(l~(l.l3’
- IW EC’s,,
h.40 * 0. IO I91 I 17 E S.hl & 0.20 309 * 23 6.28 + (12 1 2(12”k31 F &IX f(l.12
3lXrt34 ss*ll Xf I7
h.2(f+O.l7 5.46+(f.l5
Xhf21)
4.7()f().l2~
h.30 f 0. I (Y
a Maximal effect in mg force per ms of tissue dry weight. ” Not determined. The data WV means + S.E.M, trf six IO eight experiments fclr control and test tissues. P < il.05 cnmp;trcd ta cnntrnl.
47
* Central . . .
? 8
1
I
7
6
5
4
3
676543
Hiztamme
Fig. 3. The effects of phorbol 12,13-diacetate (PDA) and ouabain (left panel) or amiloride (right panel) on the concentralion-effect curves for histamine in guinea-pig isolated trachea. Two succesive concentration-effect curves to histamin.: were generated. Initial curves (control) are not shown. Second curves were made in the absence (control) or presence of ouabain (OUA), amiloride (AMI), PDA, ouabain plus PDA or amiloride plus PDA as indicated. The results are presented as percentages of maximal responses obtained in the
initial
curves and are
means
of six lo eight
(fig. 3, right). E,,, values of histamine were 324 + 24 mg/mg (n = 6) in time-matched controls, 175 + 28 mg/mg in amiloride-treated tissues (P < 0.05 VS. control; n = 6). and 201 + 27 mg/mg in amiloride plus PDA-treated tissues (P < 0.05 vs. amiloride-treated tissues; n = 6). When tracheal strips bathed in a Ca’+-free solution were challenged with acetylcholine (1 mM), histamine (I mM) or 5hydroxytryptamine (0.1 mM), only the contractions due to 5-hydroxytryptamine were depressed. Responses to acetylcholine, histamine and 5hydroxytryptamine in a Ca *+-free medium were inhibited further by incubation with ouabain (10 PM), amiloride (0.1 mM), a K+-free medium or a low Na+ solution. Ouabain failed to depress the contraction produced by acetylcholine in Ca”+-free solution (fig. 4). amiloride
PDA 5 /LM AMI 0 1 mM AMI + PDA
3.3. Effects of ouabain and amiloride on skinned tracheal preparatiom
experiments;
S.E.M. shown by vertical bars. The abscissae indicate the concentrations of the spasmogensas -log molar (MI concentration.
Segments of skinned trachea contracted in a concentration-related manner in response to Ca*+. The second concentration-effect curve for Caz+ obtained in the same preparation was slightly displaced to the left ( -log EC5,, for first curve was 6.31 f 0.03 vs. 6.44 + 0.04 for the second curve; n = 5; P < 0.05) with a small depression of its maximum (I 1.2 _t 5.6%) compared to the initial concentration-effect curve. Guabain (IO PM) and amiloride (0.1 mM) had _r~ effect on the sensitivity and maximum responses of skinned strips to Ca*+ (data not shown). The acetylcholine (100 PM) challenge terminating each experiment produced no tension increase.
4. Discussion 2
120 -
5-hydroxytryptamme
00
40
0 Contrul
AMI OUA 10 1rM ” I mM
K’frrr
Low N.,-
Fig. 4. The influence of ouabain (OLJA), amiloride (AMI). K’-free solution and low Na* (25 mM) solution on the contractile responses of the guinea-pig trachea to acetylcholine (I mM), histamine (I mM) or S-hydroxytryptamine (0.1 mM) in Ca*‘-free medium. Two succcssive challenges were generated. First challenges in normal CaZ’ (2.5 mM) PSS are not shown. Second challenges were in a &‘-free (EGTA 0.1 mM) solution in the absence (control) or presence of additions. Responses are presented as percentages of Ihat in Ihc first challenge. Column heights indicate the mean of six to eight experiments and vertical lines indicatethe S.E.M. * P < 0.05 compared to first challenge in PSS (Ca*’ 2.5 mM). ’ P < 0.05 compared to second challenge in a &‘-free medium.
The purpose of the present study was to determine whether interventions that affect membrane Na’ transport alter airway responses to spasmogens in vitro. Ouabain or external K+ removal, which inhibit Na+/K+-ATPase (Fleming, 1980), amiloride, which inhibits Na’ entry, Na’/H+ exchange and Na+/Ca’+ exchange (Reuter et al., 1973; Bcnos, 1981; Bova et al., l988), and reduction in trans-sarcolemmal Na+ gradient by exposure to a Nat-depleted medium were used. All these interventions, except amiloride produced a transient contraction followed by relaxation then return to baseline. Amiloride caused a sustained contraction. The mechanisms underlying these effects could involve, depending on the agent, Na+ loading, intracellular Ca*+ accumulation and other changes which have been reported and reviewed in the literature (Souhrada et al., 1981; Kawanishi ct ab, 1984; Knox et al., 1990). The spasmogens were selected to produce tracheal contraction predominantly due to either cxtracellular
us
qbnses
(3~ intracellular
to C&‘l.‘
~~~~ 41~ Ii;ct
etlap
Gil-’ ’
dCil!X.
‘I‘IIUS. rc-
(in Ca- +-free. K +-depolarizing
;trc the conscqucncc of cxtraccIhkIr
thmuph
voltage-dependent
Cal’
S~hl-
CO’ ’
channels
choline, histamine and S-hydroxytryptamine were generated in the absence of extraccllular Ca’+, ouabain did not affect acetylcholine-induced effective
to depressing
spasm but was as
responses to histamine
hydroxytryptamine
recuptors generate inositol I.6triphosphate which is respc>nsible for intracellular Ca’ + mobilization. and diac~;lglycer~~l which activates protein kinase C
others.
(S&amn and Grunstein. lY8Y: Rodgcr and Small. IWI ). Membrane depolarization. increase of intraccl-
Itoh, IYX4). An effect of ouabain on Ca’ ’ rclcase from sarct,plasnlic reticulum has been shown for vascular
lular Ca2 - and activation c>f protein by spasmogens may result. through
kiniWZ
C
different
nisms. in alterations of the trans-membrane ent. Na ‘-K * ATPase activity, and the Na
produced mccha-
Na ’ gradil /Ca’ + and
as in PSS. These
and S-
cpucd by dc~~larization (Foster et al.. 1YX.t: Rodgcr. lvzi7). hn clmtrast, agonists (acetylcholine. histamine. 5-hydn~qqryptaminc) that activate specific membrane
results
suggest
that ouabain may interfere with the intracellular Ca” rclcasc triggered by certain spasmogens but not by This may bc cxpkined by the cxistcncc of different intracellular Ca’ ’ stores available for diffcrcnt spasmogens. as previously shown by others (Ito and
smooth muscle (Lamb et al.. 1088). Results from the experiments with skinned
trachea
rule out the possibility that ouabain interfered
with the
intracellular contractile mechanism. Therefore, the inhibitory effect of ouabain has to be ascribed to the role
Na ‘/H + exchange systems (Schramn and Grunstcin, IYSY). Therefore. interventions which inhibit mem-
of intracellular
hranc
movements (Van Breemen et al.. 1070: Blaustein,
MI
+
transport could affect tracheal rcsponscs to
spilsmOgCIlS.
Ouahain (0.1 PM) has hccn reported to increase sensitivity of intact guinea-pip trachea to methacholinc but it dccrcascd the sensitivity to KC.1 (Racburn and I-cdan. 1’159). Ouabain did not alter methacholinc- and KCI-induced rcsponscs in cpithclium-denuded guincapig trachea (Raehurn and Fedan, IYSY). This indicates that integrity of the cpithclium is important to the effects of ouabain. All expcrimcnts in the present study were carried out in intact guinea-pig trachea. When the effect of a concentration of ouabain (10 PM) sufficient to fully inhibit Na’.K’-ATPase in guinea-pig trachea (Souhradd ct al.. IYXI) was studied on the rcqnmses to a variety ot spasmogens, it was found that only contractions due to histamine and S-hydroxylryptamine
were depressed while those due to CaCI , depolarizing so!ution). KC1 and ace&
!in Ca-“-free
choline remained unaffected. In iso,htcd human bronchi.
Black et al. (IYX4)
re-
ported that pretreatment with a high concentration of ouabain (SO PM) markedly attenuated the response :o KCI. carbachol and histamine but Knox et al. (1900) found that ouabain (IO PM) did not a!ter methacholine-induced responses. These results are in contrast with those in vascular smooth muscle where inhibition of the Na ‘.K’ pump potentiates contractions clicitcd by a variety of activating agents (Van Breemen et al.. lY7Y)_ The mechanism underlying the inhibitory effect of ouabain on airway responses to spasmogens in vitro is not clear. Interference with Ca” entry through voltage-dependent channels is ruled out since CaCl,- and KCI-induced responses are not affected. Moreover, the effects of ouabain and those of Ca’+ entry channel blockers on tracheal responsiveness to spasmogens arc not equivalent (Foster et al., 19X4; Advenier et al., 1YW this study). When maximal responses to acetyl-
Na + as modulator
of intracellular
Ca” IYXY).
Removal of external K’ also inhibits Na’/K+ATPasc but its clectrophysiological effects arc different from those observed after ouahain !Souhrada
ct al.,
19x1), Exposure to a K +-free solution dcprcssed responses to acetylcholine. tryptamine
the and S-hydroxy-
histamine
in PSS and C$+-free
solution. We have not
found other reports in the literature on these effects of K ‘-free media. The mechanism of the inhibitory effect of reduced external K’ is uncertain but considerations similar to those for ouabain are probably valid in this
rcspcct . Amiloride
depressed the responses to acetylcholine.
histamine
and S-hydroxytryptamine
Ca”-free
solution
in both
PSS and
(this study). These findings
are in
agreement with those of Knox et al. (IYYO) showing that amiloride (0.1-l mM) inhibited conlractions of bovine trachea produced by carhachol and histamine. Souhrada et al. (lYX8) reported that amiloride (IO or.M) prevent& contractile and electrical responses to spccific antigen challenge in isolated guinea-pig trachea. Unfortunately amiloride is not a selective pharmacological tool. Thus, amiloridc inhibits a Na + entry channel (Berms lY81). the Nat/H+ antiport (Benos, 1981) and Na’/Ca’+ exchange (Debetto et al. 1987) and voltage-dependent Ca’+ channels (Klcyman and Cragee. IY%H). The lack of effect of amiloride (0.1 mM) on CaCl,- and KCI-induced spasm rules out a mechanism rclatcd to inlcrfcrcncc of Ca’+ entry through voltagcdependent Ca’+ channels in this preparation. Amiloride may also inhibit protein kinase C activity (Besterman et al., lYt(S). PDA, an activator of protein kinase C (Nishizuka, lY84), partly re*lersed the inhibition produced
by amiloridc
(0.1
mM)
on histamine-induced
contraction (this study). This indicates that part of the effects of amiloride could be related to inhibition of protein with
kinase C. The a
greater
use of analogues
selectivity
for
the
of amiloride
Na ‘/H
+
and
-lY
Na +/Ca’+ exchangers Wigne ct al., 1983; Kaczorowski ct al., 1989) may help to prccisc the importancc of these systems in agonist-induced contraction of airways smooth muscle. Exposure to a low-Na + solution also depressed rcsponses to acetylcholine, histamine and S-hydroxytryptamine in PSS and Ca’+-free medium but KCI-in-
to dctcrminc
precisely
which
ccrrtijo. J.. J.S. Dixon.
Drhetto.
mcchanismb)
solution was simi-
mogens. In conclusion, interventions that affect membrane Nat transport dcprcsscd contractile rcsponscs to physiological substances of some importance in asthma.
hy emiloride. Fleming.
C’.I.C’.Y.T.
(Ministerio
in part
de Industria
hy Grunt
y Energia)
FARYII-6X0
Foster and R.C. Small.
from
Spain. The authors
thank for the v;rluahlC technical assistance of Mr. P. Santamitrin.
X-ItHI
from guinea-pig
F. Carpcnedo
IYXO. The
rlectrogrnic
Pharmacol.
20, 120.
Toxical. RI‘.
Sm;rll and A.H.
action of potassium chloride macol. XI). 5.53. Foster, R.W.. B.I. Okpalugo
lYX7. Inhihi-
in smooth
significance.
Weston.
and R.I’.
Chcalis. Br. J. Pharmacol.
Ann.
Rev.
IYX3. The spasmogenic
Small.
Br. J. Phar-
10X4. Antagonism
in guinea-pig
of
isolated tra-
Xl. 490.
10X4. The roles of stored calcium in contractions
trachC;ll smooth muscle produced hy elCctriC;ll stimulation.
ncetylcholinc
and high K
‘,
Br. J. PharmaCol. X.3, 6h7.
Javaid. A.. M.J. C’ushley and M.F. on bronchial 4%.
reactivity
G.J..
M.L.
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to histamine Garcia.
Noble and II. RKUlKr
(Oxford
University
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K.
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Baha
T.R. and E.J. Cragor,
19X4.
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inhaled ouahain
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on hrolrchial
in the
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Tattersficld
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p. (6
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KX-
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Kawanishi.
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on lension
pl;~smalcmm;d mcmhranc
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