Abstract The effects of ouabain, an inhibitor of the plas-malemmal Na+/K+-ATPase activity, were examined in hu-man isolated bronchus. Ouabain produced concentration-dependent contraction with –logEC50=7.16±0.11 and max-imal effect of 67±4% of the response to acetylcholine (1 mM). Ouabain (10 µM)-induced contraction was epithe-lium-independent and was not depressed by inhibitors ofcyclooxygenase and lipoxygenase, antagonists of muscarinic,histamine H1-receptors and α-adrenoceptors, or neuronalNa+ channel blockade. The inhibition of ouabain contrac-tion in tissues bathed in K+-free medium, and the inhibitionby ouabain of the K+-induced relaxation confirm that thecontractile action of ouabain is mediated by inhibition ofNa+/K+-ATPase. Furthermore, depolarization (16.4±0.9 mV)was observed in human isolated bronchus by intracellularmicroelectrode recording. Ouabain (10 µM)-induced con-tractions were abolished by a Ca2+-free solution but not by blockers of L-type Ca2+ channels. In human culturedbronchial smooth muscle cells, ouabain (10 µM) produceda sustained increase in [Ca2+]i (116±26 nM) abolished inCa2+-free medium. Incubation with a Na+-free medium oramiloride (0.1 mM) markedly inhibited the spasmogeniceffect of ouabain thus suggesting the role of Na+/Ca2+ ex-change in ouabain contraction while selective inhibitorsof Na+/H+-antiport, Na+/K+/Cl–-antiport, or protein kinaseC had no effect. Ouabain (10 µM) failed to increase inosi-tol phosphate accumulation in human bronchus. Ouabain(10 µM) did not alter bronchial responsiveness to acetyl-choline or histamine but inhibited the relaxant effects of

isoprenaline, forskolin, levcromakalim, or sodium nitro-prusside. These results indicate that ouabain acts directlyto produce contraction of human airway smooth muscle thatdepends on extracellular Ca2+ entry unrelated to L-type chan-nels and involving the Na+/Ca2+-antiporter.

Keywords Ouabain · Airway smooth muscle · Humanisolated bronchus · Na+/K+-ATPase · Inositol phosphates ·Intracellular free [Ca2+] · Membrane potential

Introduction

The existence of an active electrogenic Na+ pump has beendemonstrated in airway smooth muscle (Souhrada et al.1981), and its inhibition by ouabain, a Na+/K+-ATPase in-hibitor, results in contraction of airway smooth muscle ofdifferent animal species both in vitro (Souhrada et al.1981; Chideckel et al. 1987; Ortiz et al. 1991) and in vivo(Marco et al. 1968; Agrawal and Hyatt 1986).

In humans, a high salt diet augments bronchial reactiv-ity probably due to increased circulating concentrations ofendogenous inhibitors of plasmalemmal Na+/K+-ATPase(De Wardener et al. 1981). This endogenous substance(s)has been identified as indistinguishable of ouabain (Ham-lyn et al. 1991). Ouabain produces spasm of human air-ways in vitro (Chideckel et al. 1987; Knox et al. 1990), itcan evoke bronchoconstriction in asthmatics and in-creases methacholine reactivity in patients with allergywithout enhancing reactivity to methacholine or histaminein control subjects (Gentile and Skoner 1997). However,the mechanisms underlying ouabain-induced contractionand the effects of ouabain on the responses to contractileand relaxant agents have not been investigated in detailfor human airways. Therefore, the aim of the present studywas to examine some mechanical, biochemical and elec-trophysiological effects of ouabain in human isolatedbronchus and its influence on the bronchial responses todifferent spasmogenic and relaxant agents.

Julio Cortijo · Benjamín Sarria · Manuel Mata ·Emmanuel Naline · Charles Advenier ·Esteban J. Morcillo

Effects of ouabain on human bronchial muscle in vitro

Naunyn-Schmiedeberg’s Arch Pharmacol (2003) 368 : 393–403DOI 10.1007/s00210-003-0818-0

Received: 27 June 2003 / Accepted: 3 September 2003 / Published online: 15 October 2003

ORIGINAL ARTICLE

J. Cortijo · B. Sarria · M. Mata · E. J. Morcillo ()Departament de Farmacologia, Facultat de Medicina i Odontologia, Universitat de València, Avda. Blasco Ibáñez 17, 46010 Valencia, SpainFax: +34-96-3864622,e-mail: esteban.morcillo@uv.es

E. Naline · C. AdvenierFaculté de Médecine Paris-Ouest, UFR Biomédicale des Saint Pères and Centre Hospitalier de Versailles, 78157 Le Chesnay, France

© Springer-Verlag 2003

Materials and methods

Preparation of human tissues for pharmacomechanical experi-ments. Lung tissue was obtained from patients who were undergo-ing surgery for lung carcinoma. None of the patients had a historyof asthma. Experiments were approved by local ethics committee.After the resection of one or more lung lobes, a piece of macro-scopically normal tissue was immersed in physiological salt solu-tion (PSS, composition in mM: NaCl 118.4, KCl 4.7, CaCl2 2.5,MgSO4 0.6, KH2PO4 1.2, NaHCO3 25.0, glucose 11.1) at 4°C andtransported to the laboratory. Segments of bronchus were dissectedfree from parenchymal lung tissue and preparations cut (3–4 mmlength × 3–4 mm internal diameter) as previously described (Sarriaet al. 2002). Preparations were used immediately for experimentsto avoid overnight cold storage of tissues since Na+/K+-ATPase ac-tivity is inhibited by cooling (Souhrada et al. 1981; Ortiz et al.1991).

Bronchial rings were suspended on tissue hooks in 10 ml organbaths containing PSS, gassed with 5% CO2 in O2 at 37°C (pH 7.4).Each preparation was connected to a force displacement transducer(Grass FTO3) and isometric tension changes recorded on a Grasspolygraph (model 7P). The preparations were equilibrated for60–90 min with changes in bath PSS every 20 min before any phar-macological intervention occurred. A tension of 2 g was imposedat the beginning of the equilibration period, and a stable restinglevel of tone (approximately 1.2 g) was present at the end of thistime. In all experiments, human bronchi were first contracted withacetylcholine (ACh, 1 mM) and then relaxed with theophylline (3 mM) to obtain reference values for maximal contraction and re-laxation of the preparation. These challenges do not alter the sub-sequent responsiveness of the tissue (Sarria et al. 2002).

Assessment of the effect of ouabain on spontaneous bronchial toneand its modification by drugs and other interventions. A cumula-tive concentration-response curve for ouabain (10 nM–100 µM)was obtained. A near-maximal concentration of ouabain (10 µM)was selected for further experiments. This concentration is withinthe range of those used in previous studies of the effect of ouabain onhuman airways in vitro (Chideckel et al. 1987; Knox et al. 1990)and produces complete inhibition of Na+/K+-ATPase in guinea-pigtrachea (Souhrada et al. 1981; Rhoden et al. 2000). The time courseof responses to ouabain (10 µM) or its vehicle was monitored for60 min in test and time-matched control tissues respectively.

To examine whether the action of ouabain was mediatedthrough the release of mediators or neurotransmitters, some testtissues were subjected to epithelial removal (confirmed by histo-logical examination) or treated with indomethacin (2.8 µM; a cyclo-oxygenase inhibitor), zileuton (10 µM; a lipoxygenase inhibitor), amixture of atropine, phentolamine, and mepyramine (each at 1 µM,antagonists of muscarinic, α-adrenoceptor, and histamine H1 re-ceptors, respectively), compound 48/80 (100 µg/ml–1; to depleteendogenous histamine), or tetrodotoxin (1 µM; a neuronal Na+ chan-nel blocker). In these experiments, the response of test tissues toouabain was obtained in epithelium-denuded preparations or after30 min preincubation with, and in the presence of, the relevant drugtreatment, with the exception of compound 48/80 where two con-secutive 30-min challenges, each at the mentioned concentration,were carried out before ouabain administration.

To study the source of activator Ca2+, the influence of extracel-lular Na+ and K+, and the role of plamalemmal Na+-coupled trans-port mechanisms in the contractile response to ouabain, we exam-ined the response of test tissues to ouabain (10 µM) in the presenceof inhibitors of Ca2+ entry through L-type channels (verapamil 1 or10 µM, nifedipine 1 µM, and diltiazem 1 µM), Na+/Ca2+ antiporter(amiloride 100 µM), Na+/H+ antiporter (dimethylamiloride, DMA,30 µM), Na+/K+/Cl– cotransporter (bumetanide 10 µM), and pro-tein kinase C (staurosporine 1 µM, calphostin C 1 µM). In theseexperiments, the response of test tissues to ouabain was obtainedafter 30-min preincubation with, and in the presence of, the rele-vant drug treatment. The ouabain-induced contraction was alsomeasured in preparations bathed in Ca2+-free, Na+-free or K+-free

PSS. The Ca2+-free PSS was prepared by substituting EGTA (0.1 mM) for CaCl2. The Na+-free PSS was prepared by replacingNaCl by isosmolar sucrose and using tris-hydroxymethyl-aminomethane (5 mM) instead of NaHCO3 as a buffer; this solution wasbubbled with O2 and the final pH was adjusted to 7.4 by addingHCl. The K+-free PSS was prepared by removing KCl from thePSS and replacing KH2PO4 with NaH2PO4. Test tissues were ex-posed to the Ca2+-free or K+-free solutions for 30 min before andthroughout the ouabain challenge. In the case of the Na+-free me-dium, the preincubation time was increased to 60 min. The ouabain(10 µM)-induced contraction was also obtained in a PSS contain-ing tris-hydroxymethyl-amino methane (5 mM) but the normal Na+

concentration to assure that the effects observed in the Na+-freePSS were not due to a depressant effect of the buffer. In some ex-periments with Ca2+-free PSS, Ca2+ readmission was effected after30 min of ouabain challenge. In further experiments, the responseof test tissues to ouabain (10 µM) was measured after 30-minpreincubation with, and in the presence of, Ca2+-free PSS contain-ing both EGTA (0.1 mM) and ryanodine (10 µM). Also, the influ-ence of ouabain (10 µM, 30-min preincubation with Ca2+ free PSS)on the subsequent contraction to ryanodine (10 µM) in the contin-ued presence of Ca2+-free PSS was examined. Additional experi-ments explored the responses to K+ readmission (KCl 30 mM) intissues previously bathed for 30 min in K+-free PSS and contractedor not with ACh (50 µM). Where appropriate, the contractile re-sponses to ACh (1 mM) were examined for comparison with theouabain (10 µM) responses.

Assessment of the effects of ouabain on bronchial responses tocontractile and relaxant agents. Two successive, cumulative con-centration-effect curves were constructed for ACh (1 nM–1 mM)or histamine (1 nM–1 mM). After the construction of the first ofthese curves, the preparations were washed and, after recovery ofbaseline, tissues were allocated randomly in equal numbers to test(incubation with ouabain 10 µM for 30 min) or time-matched con-trol groups, and a second concentration-effect curve was generated.

In separate experiments, bronchial preparations were contractedwith ouabain (10 µM) or an equieffective concentration of hista-mine (3–10 µM). Once the contractile response reached a plateau,cumulative relaxation-response curves were constructed for iso-prenaline (1 nM–10 µM), forskolin (10 nM–10 µM), levcromakalim(0.1–30 µM), or sodium nitroprusside (0.3 µM–0.3 mM).

Measurement of the effects of ouabain on inositol phosphate accu-mulation. Total inositol phosphate accumulation was determinedas previously outlined (Cortijo et al. 1997). In short, cryostored hu-man bronchi (–80°C, in fetal calf serum containing 1.8 M DMSO;Sarria et al. 1995) from 3–5 patients were rapidly thawed in a 37°Cwater bath and rinsed in a large volume of PSS to eliminate theDMSO. They were cut into small fragments (about 1 mm2) and thepool, weighing a total of ~10 g was washed in PSS and incubatedin 25 ml PSS containing 50 µCi of myo-[3H]-inositol for 4 h at37°C. After this incubation, the tissue was washed twice with PSS.Aliquots of washed tissue (1.5–2 g) were placed in 3 ml final vol-ume of PSS and preincubated at 37°C for 30 min. Just before stim-ulation, 30 µl of LiCl was added (final concentration 20 mM). Thesamples were then challenged with 30 µl PSS (control), acetylcholine(0.1 or 3 mM; the latter served as a reference value of 100%) orouabain (0.1, 1 or 10 µM) for 30 min at 37°C. Stimulation of ino-sitol phosphate accumulation was stopped by addition of 3 ml ofice-cold mixture of chloroform/methanol/HCl 12 M (100:200:4,v/v/v) and vigorous shaking. The samples were centrifuged (4,000 g)for 10 min at 4°C and the aqueous phases were brought to pH 4 andstored at –20°C until analysis. The separation of inositol phos-phates was performed by an HPLC ion-exchange system (lineargradient of potassium phosphate 1 M, pH 3.7), and the radioactiv-ity was measured in a Flow-One on-line radioactivity detector(Packard, Meriden, USA).

Measurement of the effect of ouabain on [Ca2+]. These experi-ments were performed using primary cultures of human bronchialmuscle obtained from lung resection as indicated above. Previous

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experiments have demonstrated that airway smooth muscle cells inculture possess a functional Na+/K+-pump and various plasmalem-mal ion transport mechanisms (Rhoden et al. 2000). The airwaysmooth muscle was dissected with the aid of a binocular-operatingmicroscope (×10 magnification) under sterile conditions in Dul-becco’s modified Eagles’s medium (DMEM) supplemented with10% fetal calf serum (FCS), 2 mM glutamine, penicillin (100 U/ml–1),streptomycin (100 µg/ml–1) and amphotericin B (2.5 µg/ml–1). Oneto two grams of wet tissue was minced and washed in supplementedDMEM. The tissue slurry was centrifuged at 150 g for 5 min at4°C, and the pellet was resuspended in 10 ml of DMEM containing640 U/ml collagenase (type IV, Sigma) and 10 U/ml elastase (type I,Sigma), and enzymatically digested for 90 min in a shaking bathat 37°C. The cell suspension was filtered and centrifuged at 150 gfor 10 min, resuspended in DMEM/Ham’s F12 medium supple-mented as indicated above, counted in a hemocytometer, and via-bility assessed by trypan blue exclusion (>95%). Cell suspensionswere plated in 25 cm2 flasks at 37°C in a humidified atmosphere of5% CO2 in air, with an exchange of spent medium for fresh at 48-hintervals. Cells were subcultured after reaching confluency. Sub-culturing was performed up to 4–5 passages where cells show a sta-ble α-actin expression examined with a monoclonal antibody againstα-smooth muscle actin (1:100 dilution; Sigma) using the avidin-bi-otin-peroxidase method; >95% of the cells were positively stained.

[Ca2+]i was measured by epifluorescence microscopy (Spectra-master System, Perkin Elmer, Life Sciences, Cambridge, UK) us-ing the fluorescence Ca2+ indicator dye fura-2 as previously out-lined (Maxwell et al. 1998). Airway smooth muscle cells were seededonto 22-mm square coverslips and housed in sterile culture plates(33 mm × 10 mm) (Falcon Primaria, Becton Dickenson & Co., Frank-lin Lakes, NJ, USA). Cells were left overnight in 2 ml DMEM/F12 to allow them to attach and flatten onto the coverslip. Cellswere loaded with fura-2 acetoxymethyl ester (5 µM in DMEM/F12culture medium for 50 min at 37°C) and washed three times with1.5 ml of buffer (composition in mM: NaCl 137, KCl 5.4, CaCl22.5, MgSO4 1.47, D-glucose 11, KH2PO4 1.47, Na2HPO4 2.8,NaHCO3 1.4, and BSA 0.25% w/v (imaging medium). The cover-slip with attached cells was then placed in a circular thermostated(37°C) chamber (Ambient Temperature Controlled PerfusionChamber Module, Perkin Elmer) bathed in 0.5 ml of imaging me-dium, and mounted on the stage of an inverted microscope(Eclipse TE300, Nikon Co., Tokyo, Japan). The effects of drugs on[Ca2+]i were examined in fura-2 loaded cells, which had been equi-librated for 15 min in calcium containing imaging buffer (0.5 ml).Following establishment of a stable [Ca2+]i baseline, ouabain (10 µM)or histamine (10 µM) was added and the changes were monitoredfor 3 min. This concentration of histamine was selected as equief-fective with ouabain (10 µM) in contracting human isolatedbronchus. In additional experiments, the effect of ouabain (10 µM)was assessed in Ca2+-free, EGTA (0.1 mM), PSS. In another set ofexperiments in PSS, cells were challenged with histamine (10 µM)10 min after addition of ouabain (10 µM).

Estimation of [Ca2+]i was achieved by ratiometric analysis offura-2 fluorescence images elicited by excitation at 340 nm and380 nm in single cells. Background levels of fluorescence at eachexcitation wavelength were determined in cell free areas and sub-stracted for each experiment. Calculation of [Ca2+]i from theF340:F380 ratio was according to Grynkiewicz et al. (1985). Maxi-mum and minimum fluorescence intensities were obtained with0.1% Triton X-100 and 10 mM EGTA in 2 M Tris-HCl, pH 8.5, re-spectively as previously outlined (Cortijo et al. 1997).

Electrophysiological studies. The tissue bath used and other tech-nical aspects have been previously described (Cortijo et al. 1997).The mucosal membrane covering the luminal surface of the bronchuswas carefully removed with a cotton bud and the tissue was set upfor the simultaneous recording of intracellular electrical activityand mechanical changes. Microelectrodes (30–50 MΩ; tip poten-tial less than 5 mV) were filled with 3 M KCl and were used inconjunction with a WPI Intra 767 amplifier. One or two prepara-tions from each patient (a total of 15 preparations from 8 patients)were examined. In 7 patients (each one yielding one preparation),ouabain (10 µM) was added to the superfusing physiological salt

solution and changes in membrane potential and tension weremonitored until dislodgement of the microelectrode from the cell.

Drug sources and statistical analysis of results. The followingdrugs were used: acetylcholine chloride, atropine sulphate, bumet-anide, dimethylamiloride hydrochloride (DMA), dimethylsulphox-ide (DMSO), ethyleneglycol-bis-(β-amino-ethyl-ether)-N-N’-tetra-acetic acid (EGTA), forskolin, histamine hydrochloride, indometh-acin, isoprenaline, mepyramine maleate, ouabain, phentolaminehydrochloride, sodium nitroprusside, staurosporine, (each fromSigma), amiloride (a gift from Merck, Sharp & Dohme), diltiazemand zileuton (gifts from Laboratory Dr. Esteve, Barcelona, Spain),nifedipine (a gift from Bayer), (±)-verapamil hydrochloride (Bio-sedra-Knoll), levcromakalim (a gift from SmithKline BeechamPharmaceuticals, UK), and ryanodine (Calbiochem). Other chemi-cals used were of analytical grade (E. Merck; Panreac). Stock so-lutions of bumetanide, indomethacin, nifedipine, ryanodine andzileuton were prepared in absolute ethanol. Stock solutions of fors-kolin and calphostin C were prepared in DMSO. Fura-2 was pre-pared from a 1 mM stock solution in DMSO. Other substanceswere dissolved in PSS just before use. Nifedipine, staurosporineand calphostin C solutions were protected from light exposure; iso-prenaline solutions contained ascorbic acid. Vehicle controls (drugsolvent only) were run in parallel; no significant vehicle effectswere observed. Drug concentrations are expressed as final bathconcentrations of the active species. Water purified on a Milli-Qsystem (Millipore Iberica, Madrid, Spain) was used throughout.Isosmolarity of solutions was confirmed with an Osmomat 030(Gonotec).

Contractile and relaxant responses are expressed in absolutevalues (g or mg) and/or as a percentage of the initial reference con-tractile (ACh, 1 mM) and relaxant (theophylline, 3 mM) responses,as indicated. The effective concentration 50% (EC50) of spasmo-gens and relaxants was calculated by nonlinear regression analysisof log concentration-effect curves (Prism, GraphPad software).Data are presented as means±SEM. The number of experiments isexpressed as ‘n/p’ where ‘n’ represents the number of preparationsexamined, and ‘p’ the number of patients from which those tissueswere derived. Statistical analysis of the results was performed byanalysis of variance followed by Bonferroni multiple comparisontest (GraphPad software). Differences were considered significantwhen P<0.05.

Results

Contraction of human bronchus by ouabain

Ouabain (10 nM–100 µM) applied cumulatively to humanisolated bronchus caused concentration-dependent tensiondevelopment (Fig. 1). The maximal effect of ouabain was1.21±0.16 g, which was equivalent to 67.3±4.2% (n/p=9/5)of the contraction elicited by ACh (1 mM). The –logEC50for ouabain was 7.16±0.11 (n/p=9/5). A concentration ofouabain producing near-maximal effect (10 µM) was se-lected for further experiments. The onset of tension devel-opment in response to ouabain (10 µM) occurred within1 min of challenge and maximum tension was achievedafter 20–25 min (Fig. 2). This tension was maintained forup to 60 min, the longest time-span investigated. Responsesto ouabain were reversible following washout and repro-ducible contractions could be elicited at 30-min intervals(data not shown).

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Role of epithelium and endogenous mediators inouabain-induced contractions

Removal of the epithelium did not alter the contractionelicited by ouabain (10 µM; Table 1). Tissue incubationwith either indomethacin (2.8 µM) or zileuton (10 µM) for30 min had no effect on basal tone and did not modify thesubsequent contractile response to ouabain (10 µM; Table1). The initial exposure of bronchial strips to compound48/80 (100 µg/ml–1) induced a contraction equivalent to53.8±6.3% of that evoked by ACh (1 mM; n/p=6/4), but

the response to the second challenge with 48/80 was only12.3±5.2% (n/p=6/4) of the response evoked by ACh. Thecontractile response to ouabain in tissues twice-chal-lenged with compound 48/80 did not differ from that seenin time-matched control tissues (Table 1). Similarly, thecontractile response to ouabain was unaltered by tissuetreatment with a mixture of atropine, phentolamine, andmepyramine (each at 1 µM) or by treatment with tetrodo-toxin (1 µM; Table 1). Taken together, the results fromthese experiments indicate that the contraction producedby ouabain results from a direct action on bronchial smoothmuscle and is not likely mediated by the release of neuro-transmitters or mediators.

Influence of extracellular Ca2+, Ca2+ channel blockersand ryanodine on ouabain-induced contraction

Baseline tension fell when normal PSS was changed to aCa2+-free PSS containing 0.1 mM EGTA (35.3±2.8%;n/p=26/14). Contractions to ouabain (10 µM) elicited after30 min of incubation and in the presence of a Ca2+-free,0.1 mM EGTA, PSS were virtually abolished (10 out of14 preparations from 7 patients) although a residual con-tractile response (between 15 and 25% of ACh 1 mM) wasobserved in some preparations (21.5±1.9%; n/p=4/3);mean values for all experiments are presented in Fig. 2and

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Fig. 1 Log concentration-response curve for the contractile actionof ouabain in human isolated bronchus. Points indicate mean ±SEM from n/p=7/4

Fig. 2 The spasmogenic effect of ouabain (10 µM) in human iso-lated bronchus: its time course and the effects of some drugs andmodified physiological salt solutions (PSS). Curves for ouabainwere obtained in the absence (time-matched control tissues) and inthe presence of verapamil, staurosporine, amiloride, Ca2+-free (0.1 mM EGTA) PSS, or Na+-free PSS. Points indicate means ±SEM of number of experiments indicated in Table 1. A significantdifference (P<0.05) was found for tissues treated with Ca2+-freemedium, Na+-free solution and amiloride with respect to control(i.e., ouabain alone curve). ACh acetylcholine

Table 1 Analysis of the contractile action of ouabain (10 µM) inhuman isolated bronchus. n number of preparations, p number ofpatients from which the tissues were derived, PSS psychologicalsalt solution, DMA dimethylamiloride, ACh acetylcholine, EGTAethyleneglycol-bis-(β-amino-ethyl-ether)-N-N’-tetraacetic acid

Procedure or agent Control n/p Test n/ptissues tissues

Epithelial removal 63.3± 5.4 4/3 62.7± 5.3 4/3Indomethacin (2.8 µM) 59.7± 4.3 3/3 61.6± 5.9 3/3Zileuton (10 µM) 61.2± 2.8 3/3 60.9± 3.8 3/3Compound 48/80 64.8± 3.9 6/3 67.2± 4.7 6/3Antagonist mixture 66.4± 4.3 5/4 69.4± 5.3 5/4Tetrodotoxin 63.4± 5.7 3/3 60.6± 4.9 3/3Verapamil (1 µM) 63.0± 8.3 4/3 77.7±10.2 4/3Verapamil (10 µM) 60.4± 5.9 4/3 78.0±18.7 4/3Nifedipine (1 µM) 59.8± 3.9 4/3 55.0± 6.8 4/3Diltiazem (1 µM) 63.4± 5.2 4/3 55.0± 8.8 4/3Ca2+-free PSS 67.0±10.1 14/7 8.2± 2.6* 14/7Na+-free PSS 64.0±10.0 9/5 4.3± 0.2* 5/3K+-free PSS 60.3± 4.9 5/3 11.6± 8.4* 5/3Amiloride (100 µM) 65.1± 7.4 5/3 21.2± 7.9* 5/3DMA (30 µM) 65.3± 7.8 5/3 60.3± 7.8 5/3Bumetanide (10 µM) 65.3± 7.8 5/3 63.7± 9.3 6/3Staurosporine (1 µM) 61.3± 5.8 6/3 63.6± 6.2 6/3Calphostin C (1 µM) 61.3± 5.8 6/3 60.6± 4.8 4/3

Data represent responses (mean ± SEM) to ouabain expressed as apercentage of the contractile response to ACh (1 mM). Antagonistmixture contains atropine + phentolamine + mepyramine (each at1 µM). Ca2+-free PSS contains EGTA (0.1 mM). Some of the ex-periments were performed concurrently and hence used a commongroup of control tissues*P<0.05 from the corresponding control value

Table 1. Under these conditions, readmission of Ca2+ inthe presence of ouabain resulted in immediate tension de-velopment that reached values similar to those induced byouabain in normal PSS (not shown; n/p=3/3).

Since the bronchial contraction elicited by ouabain wasdepending on extracellular Ca2+ entry, we then examined theinfluence of drugs that inhibit Ca2+ influx through L-typechannels. Verapamil (1 or 10 µM), nifedipine (1 µM) ordiltiazem (1 µM) each caused reduction of the spontaneoustone of the tissues (13.2±1.7%, 21.1±2.3%, 15.2±1.6%,and 13.5±1.4%, respectively; n/p=6/4 for each concentra-tion). Incubation with the mentioned Ca2+ channel antag-onists failed to reduce the contraction to ouabain (10 µM)in normal PSS (Fig. 2, Table 1). These results indicate thatactivator Ca2+ for ouabain contraction is not entering theairway smooth muscle cells through L-type Ca2+ channels.

To test whether ouabain utilizes activator Ca2+ releasedfrom intracellular stores, we used ryanodine to releaseCa2+ from their intracellular stores. Addition of ryanodine(10 µM) to the Ca2+-free, EGTA-containing PSS caused asmall, transient contraction; however, ryanodine did nototherwise modify the tissue tone loss induced by the Ca2+-free medium. In tissues bathed in Ca2+-free medium, pre-treatment with ouabain (10 µM) did not modify the subse-quent contractile response to ryanodine (data not shown).

The addition of ryanodine to the Ca2+-free medium didnot further depressed the residual contractile response toouabain (10 µM) observed in some tissues (21.5±1.9%and 19.4±3.4% of ACh 1 mM in the absence and presence ofryanodine, respectively; n/p=4/3 for each group, P>0.05).Therefore, intracellular Ca2+ is not apparently a main sourceof activator Ca2+ for ouabain contraction.

Influence of K+-free PSS and K+-readmission, Na+-free PSS and drugs affecting Na+ fluxes, and protein kinase C inhibitors on ouabain-induced contraction

Exposure of bronchial rings to a K+-free PSS resulted inan initial reduction of baseline tension followed by recov-ery to pre-exposure levels 30 min later (Fig. 3). This inter-vention inhibits Na+/K+-ATPase and, as expected, markedlydepressed the contractile response to ouabain (10 µM;Table 1) without affecting the contraction to ACh (responsesto ACh 1 mM were 96.5±1.22% in PSS and 89.6±1.37%in K+-free PSS; n/p=5/3). In tissues bathed in K+-free PSSfor 30 min, the readmission of K+ (KCl 30 mM) reacti-vates the Na+/K+-ATPase and evoked a transient relaxationfollowed by a rise in tension above the pre-existing level.The relaxation induced by K+ readmission (KCl 30 mM)was observed also in tissues bathed in K+-free medium for30 min and contracted with ACh (50 µM). In test tissuesthat had been treated with ouabain (10 µM), the readmis-sion of K+ evoked contraction only (Fig. 3).

Exposure to a Na+-free PSS resulted in rapid contrac-tion (peak of 89.3±6.1% of ACh 1 mM in 4–5 min; n/p=5/3) followed by relaxation and return to baseline occur-ring by 20–30 min. The transient contraction elicited byomission of extracellular Na+ was abolished in prepara-tions bathed in Ca2+-free, EGTA (0.1 mM) PSS (n/p=3/3).Contraction to ouabain (10 µM) was abolished when ob-tained in a Na+-free solution (Fig. 2, Table 1). This was not

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Fig. 3a–d Inhibition by ouabain (OUA) of the relaxation inducedby K+ readdition in human isolated bronchus bathed by K+-freephysiological salt solution. Responses of four preparations ob-tained from the same patient are illustrated. Upper traces: controltissues – note the initial contraction to ACh (1 mM), the transientrelaxation produced after exposure to K+-free medium and, 30 minlater, the relaxation produced by readdition of K+ (KCl 30 mM) ei-ther a on basal tone or b on ACh-induced tone. The initial K+-in-duced relaxation was followed by a rebound contraction in bothcases. Lower traces: test tissues – note that addition of ouabain(10 µM) failed to produce contraction but abolished the relaxationproduced by K+ readdition without preventing the subsequent con-traction (c, d). These results are representative of observationsmade in three patients

due to a non specific depressant effect of the buffer (tris-hydroxymethyl-amino methane) since ouabain (10 µM)-induced contraction was not reduced in experiments withthe same buffer concentration but normal concentration ofNa+ (66.3±4.5 and 67.8±6.2% in the absence and presenceof buffer; n/p=6/3). These results indicate that the Na+/Ca2+ exchange may be an important source of activatorCa2+ for ouabain contraction.

Amiloride (100 µM), a non selective inhibitor ofNa+/Ca2+ exchange in smooth muscle, produced a small tran-sient contraction (peak of 18.3±7.0% of ACh 1 mM, n/p=5/3, in about 10 min with baseline recovered by 25–30 min)and reduced the contraction produced by ouabain (Fig. 2,Table 1) without affecting ACh (1 mM)-induced contrac-tion (96.5±1.22% in control and 83.7±5.3% in amiloride-treated tissues, n/p=5/3). The amiloride derivative, dimethyl-amiloride (DMA, 30 µM), a selective inhibitor of Na+/H+

exchange, did not significantly changed baseline tension andfailed to depress the ouabain-induced contraction (Table 1).Bumetanide (10 µM), a selective inhibitor of Na+/K+/Cl– co-transporter, reduced baseline tension (–28.6±6.1%; n/p=6/3)but did not significantly alter the contraction produced byouabain (Table 1).

Staurosporine (1 µM), a selective protein kinase C in-hibitor, neither significantly altered the baseline tension nordepressed the ouabain (10 µM)-induced contraction (Ta-ble 1). Similar results were obtained for another selectiveprotein kinase C inhibitor, calphostin C (1 µM; Table 1).

Effects of ouabain on inositol phosphates

Ouabain failed to produce an accumulation of inositol phos-phates in the human isolated bronchus (0.37±0.03, 0.36±0.05, 0.34±0.06, 0.35±0.05 d.p.m. ×104 g–1 in the absenceand presence of ouabain 0.1, 1 and 10 µM respectively;n/p=4/4). In contrast, preparations from the same patientsresponded to ACh (3 mM) with an accumulation of inosi-tol phosphates (2.7±0.9 d.p.m. ×104 g–1, n/p=4/4, P<0.05from values showed above for control and test tissues).Pretreatment of tissues with ouabain (10 µM) did not alterthe subsequent inositol phosphate response to ACh (0.1 or3 mM; not shown; n/p=3/2).

Effects of ouabain on [Ca2+]i

Baseline values of [Ca2+]i found in this study were withinthe range of those previously reported (Cortijo et al.1997). Addition of ouabain (10 µM) produced an immedi-ate but relatively slow rise in [Ca2+]i reaching maximalvalues in approximately 40 s (Fig. 4a). Subsequently, theaugmented [Ca2+]i levels were maintained above baselinevalues at the end of the initial period of observation (3 min)and of additional observation times (10 and 30 min afterouabain challenge; not shown). This pattern of Ca2+ signalwas different from that elicited by a concentration of hist-amine (10 µM) producing a contraction equivalent to thatof ouabain. The addition of histamine resulted in a very

rapid and transient increase of [Ca2+]i (peak reached inless than 10 s) followed by a sustained phase above base-line values (Fig. 4b). The magnitude of the sustained in-crease in [Ca2+]i in response to ouabain was about 10% ofthe peak increase elicited by histamine but represented anamount of [Ca2+]i similar to the level existing in the hista-mine plateau. Preparations exposed to ouabain (10 µM) re-sponded to a subsequent challenge with histamine (10 µM)with a calcium signal similar to that observed in controluntreated preparations (not shown; n/p=3/3).

Electrophysiological studies

The resting membrane potential of the human bronchialmuscle was –45.7±1.3 mV (range between –36 mV and–52 mV, n/p=15/8). In 5 out of 15 of the examined cells,spontaneous slow and oscillatory potential changes (slowwaves) were recorded while the other cells exhibited ir-regular potential changes as previously described (Cortijoet al. 1997). The resting membrane potential in cells sub-sequently exposed to ouabain was –46.1±1.4 mV (n/p=7/7). The addition of ouabain (10 µM) to the superfusateevoked a slowly developing depolarization that reachedmaximal values of 16.4±0.9 mV (n/p=7/7). A representa-tive trace is shown in Fig. 5. These electrical changeswere accompanied by a parallel gradual increase in tissuetension (427±69 mg).

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Fig. 4 a Representative curve illustrating the [Ca2+]i response toouabain (10 µM) in human cultured airway smooth muscle cells.b For comparison, the [Ca2+]i response obtained for an equieffec-tive concentration of histamine (10 µM) is shown. Note the differ-ence between the ordinate scales. In ouabain experiments the base-line [Ca2+]i was 125±11 nM and the maximal response attained at35 s amounted 266±37 nM which represents an increment (peakminus baseline values) of 141±28 nM. The [Ca2+]i response wasmaintained and the increment at the end of the initial observationperiod was 116±27 nM. In histamine experiments the baseline[Ca2+]i was 173±13 nM and the peak response attained at 7 samounted 1,769±239 nM which represents an increment (peak mi-nus baseline values) of 1,595±237 nM. The [Ca2+]i response de-clined rapidly to a plateau of 304±17 nM which represents an in-crement above baseline (dashed line) at the end of the observationperiod of 130±18 nM. Image acquisition was made at intervals of4.37 s for ouabain and of 3.79 s for histamine. Data are mean ±SEM from a single patient where n=14 cells, and is representativeof results obtained from three patients

Effects of ouabain on the responses to spasmogens and relaxants in human bronchus

ACh and histamine each produced reproducible concen-tration-dependent contractions of human isolated bronchus.The Emax (expressed as a percentage of the peak tensionevoked by ACh 1 mM) and –log EC50 values for ACh ob-served in the second log concentration-effect curve incontrol, untreated tissues were 97.4±1.4% and 5.15±0.08respectively (n/p=5/5). The corresponding values for his-tamine were 94.6±3.1% and 5.89±0.08 (n/p=5/5). In testtissues (i.e., ouabain treated), responses to ACh and hista-mine that were obtained in the second concentration-re-sponse curve commenced from an elevated baseline. Thiselevated baseline represented the plateau contraction pro-

duced by ouabain (10 µM). Despite the increase in toneinduced by ouabain, the responsiveness (Emax) and sensi-tivity (–log EC50) of the preparations to ACh (111.2±10.8% and 4.99±0.29) and histamine (119.0±11.6 and5.62±0.24) was not significantly altered compared with theequivalent parameters measured in control tissues (n/p=5/5; Fig. 6).

To study the influence of ouabain in relaxant responses,concentration-effect curves for isoprenaline, forskolin, lev-cromakalim and sodium nitroprusside were constructed intest tissues contracted with ouabain (10 µM) while controluntreated preparations were contracted to a plateau withequieffective concentrations of histamine (3–10 µM) sincethe efficacy and potency of relaxants is affected by thepre-existing level of tone from which the relaxant re-sponses are obtained. Relative to the curves observed inhistamine-contracted control tissues, the log concentra-tion-response curves for isoprenaline, forskolin, levcro-makalim and sodium nitroprusside were depressed inouabain-treated preparations (Fig. 7) but the potency ofthese relaxants was not significantly altered (–log EC50values in control and test tissues were 7.19±0.06 and6.32±0.13 for isoprenaline, 6.46±0.06 and 6.63±0.15 forforskolin, 5.34±0.11 and 5.03±0.25 for levcromakalim,and 4.91±0.10 and 4.88±0.12 for sodium nitroprusside,n/p=5/5 in each group).

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Fig. 5 The effects of ouabain on the intracellular electrical activ-ity and tension changes in human isolated bronchus. Upper trace:membrane potential changes. Lower trace: tension changes of acontiguous segment of bronchus. All the record was taken from thesame cell. Note the impalement of cell and baseline recording ofmembrane potential. The open triangles indicate the moment atwhich ouabain (OUA, 10 µM) was added. Ouabain produced agradual depolarization and contraction. The upper trace ends withthe dislodgement of the electrode from the cell. The trace showedis representative of results obtained in seven patients

Fig. 6 The effect of ouabain on the log concentration-responsecurve of acetylcholine and histamine in human isolated bronchus.Two successive concentration-effect curves were generated withthe second curves obtained in the absence (time-matched controls)or presence (test tissues) of ouabain (10 µM). Concentration-re-sponse curves in ouabain-treated tissues started at a higher level ofcontraction compared to the corresponding control curves. Pointsare means ± SEM of values from n/p=5/5

Fig. 7 The effect of ouabain on the log concentration-responsecurve of isoprenaline, forskolin, levcromakalim and sodium nitro-prusside in human isolated bronchus. Concentration-effect curveswere generated in control untreated tissues or in test tissues treatedwith ouabain. Control tissues were precontracted with histamine ina concentration (3–10 µM) titrated to be equieffective with ouabain(10 µM). Points are means ± SEM of values from n/p=5/5

Discussion

The bronchial contraction of ouabain is unrelated to mediator release

Ouabain produced a concentration-dependent (10 nM to100 µM) contraction of human isolated bronchus. Thiscontraction was sustained (up to 60 min of monitoring)showing that ouabain can exert prolonged effects on thetone of human airways smooth muscle. The maximal re-sponse to ouabain was near 70% of that of ACh maxi-mum. These results confirm and extend other studies withouabain in human isolated airways (Chideckel et al. 1987;Knox et al. 1990). The EC50 for ouabain in human isolatedairways is close to 0.1 µM, a value consistent with data fromhuman tracheae obtained during autopsies (Chideckel etal. 1987).

The resistance of ouabain-induced spasm to atropine (1 µM, an effective concentration to block muscarinic M3receptors in human airways; Sarria et al. 2002), and phen-tolamine (1 µM, a concentration that blocks human α1-ad-renoceptors), suggests that ouabain contraction is notmediated by the local release of ACh or noradrenaline.Tetrodotoxin (1 µM, a neuronal Na+ channel blocker) alsofailed to reduce ouabain-induced contraction suggestingthat this effect was not likely related to excitatory neuro-transmitters released from intramural nerve terminals. Theineffectiveness of mepyramine (1 µM, a concentration thatblocks histamine H1 receptors) and compound 48/80 (inconcentrations shown to virtually deplete histamine storesin mast cells) to alter the ouabain contraction suggests thatrelease of histamine is not involved. The spasmogenic ef-fect of ouabain in human bronchus does not result fromstimulation of prostaglandin synthesis since indomethacin(2.8 µM, a concentration sufficient to suppress prosta-glandin synthesis by human bronchial tissue) failed to an-tagonize ouabain (Table 1). The failure of zileuton (10 µM,a concentration which inhibits 5-lipoxygenase activity inhuman tissue), to antagonize ouabain-induced spasm ofhuman bronchus (Table 1) suggests that ouabain contrac-tion does not depend on leukotriene production. Further-more, epithelial removal did not modify the action ofouabain (Table 1) indicating that ouabain is not evokingthe release of epithelium-derived factors or that epithelialfactors are not modulating the muscle Na+/K+-ATPase(Raeburn and Fedan 1989). Collectively these observa-tions suggest that the spasmogenic action of ouabain inhuman isolated bronchus is unlikely to involve the releaseof neurotransmitters and mediators from structures withinthe bronchial wall but, instead, represents a direct actionof ouabain on the smooth muscle cells.

Ouabain inhibits Na+/K+-ATPase and depolarizes human bronchial muscle

Ouabain is considered an inhibitor of plasmalemmal Na+/K+-ATPase in a number of tissues. This enzyme exists in

different isoforms which display different affinities for[K+]o and different sensitivities to ouabain (Crambert etal. 2000) but no detailed information is currently availablefor human airway smooth muscle cells. In this study, therole of Na+/K+-ATPase inhibition in the direct action ofouabain to produce contraction of human bronchus wasfirst examined by interventions modifying [K+]o. As ex-pected, the ouabain (10 µM)-induced contraction of hu-man isolated bronchus was substantially reduced in tis-sues incubated in K+-free solution, a maneuver that in-hibits the Na+/K+-ATPase activity (Souhrada et al. 1981).Also, K+ readmission after incubation of human bronchusin K+-free medium re-activates the Na+/K+-ATPase andproduces a transient inhibition of spontaneous and ACh-induced tone that was fully inhibited by ouabain (10 µM),thus confirming and extending previous work from thislaboratory (Cortijo et al. 1997). In keeping also with thismechanism of action, ouabain (10 µM) increases the so-dium content of human isolated bronchus (Cortijo et al.1997).

Since the Na+ pump is electrogenic, another conse-quence of the inhibition of Na+/K+-ATPase is depolariza-tion (Souhrada et al. 1981). The resting membrane poten-tial and the pattern of spontaneous electrical activity ofhuman bronchial muscle found in this study was similar tothose previously reported (Cortijo et al. 1997). Ouabain(10 µM) produced a slowly developing depolarization ofhuman isolated bronchus, which confirms and extends thefinding of a similar pattern and size of ouabain-induceddepolarization in guinea-pig and bovine trachealis (Souhradaet al. 1981). Since ouabain inhibits the Na+/K+-ATPase,the observed depolarization presumably results from in-tracellular Na+ accumulation due to blockade of the out-ward pump current. In addition, the opening of the Ca2+-dependent Cl–-channels that have been suggested to existin the plasmalemma of airway smooth muscle cells(Janssen and Sims 1992) may also contribute to the depo-larization induced by ouabain. The depolarization evokedby ouabain does not apparently lead to the opening of asignificant number of voltage-operated channels mediat-ing extracellular Ca2+ influx since the pharmacologicalblockade of these channels did not reduce the ouabain-in-duced contraction (see below).

Main source of activator Ca2+ utilized by ouabain is of extracellular origin

Ouabain promoted a relatively small but sustained rise of[Ca2+]i in human airway smooth muscle cells. This findingis consistent with a monophasic, slowly developing con-traction in response to ouabain. The contraction to ouabainand the [Ca2+]i increase were abolished or markedly re-duced in tissues bathed in a Ca2+-free PSS containingEGTA (0.1 mM). Such concentration of EGTA is envis-aged to chelate extracellular Ca2+ and Ca2+ bound at su-perficial sites on the plasmalemma, but to leave the intra-cellular stores of Ca2+ largely intact. Hence, our findingsindicate that the ouabain-induced contraction of human

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bronchus depends on an extracellular source or a superfi-cial pool of Ca2+ that is accessible to EGTA.

In some experiments where a residual contractile re-sponse (<25% of ACh 1 mM) was observed in Ca2+-freemedium, additional experiments with ryanodine were car-ried out. Ryanodine interferes Ca2+ mobilization specifi-cally from the sarcoplasmic reticulum in a variety of tis-sues including airway smooth muscle (Gerthoffer et al.1988). In canine and guinea-pig trachealis, ryanodine hasbeen shown to inhibit preferentially the contraction elicitedby spasmogens, which predominantly mobilize Ca2+ fromintracellular stores. We found that ryanodine (10 µM) didnot affect the residual contraction elicited by ouabain inCa2+-free EGTA (0.1 mM) PSS thus suggesting thatouabain is not utilizing Ca2+ released from ryanodine-sen-sitive intracellular stores as a relevant source of activatorCa2+ for contraction. However, the possibility of a partic-ipation of the intracellular calcium store via a calcium-in-duced calcium release phenomenon has not been ruled out(Hyvelin et al. 2000).

Increase of intracellular Ca2+ could also result fromstimulated production of inositol phosphate or inhibitionof Ca2+-ATPase in the endoplasmic reticulum. We foundthat ouabain (up to 10 µM) did not evoke an accumulationof inositol phosphates while ACh (3 mM) was effective inpreparations from the same patients. Also, we previouslyreported that vanadate, a Ca2+-ATPase inhibitor, failed toinhibit the ouabain contraction (Cortijo et al. 1997). Takentogether, these results indicate that the main source of ac-tivator Ca2+ used by ouabain is not of intracellular but ofextracellular origin.

Ouabain promotes extracellular Ca2+ entry by Na+/Ca2+

exchange and not through L-type channels

Extracellular Ca2+ may gain access to the cell -among otherpossibilities- through the voltage-operated Ca2+ channel.The presence of the L-type Ca2+ channel is well estab-lished in human airway smooth muscle (Marthan et al.1989). We examined three structurally different blockersof L-type channel (verapamil, nifedipine, diltiazem) atconcentrations sufficient to depress responses of humanisolated bronchus to histamine and KCl (Marthan et al.1989). Neither of the Ca2+ channel blockers tested de-pressed the contractile responses to ouabain in human iso-lated bronchus thus suggesting that the Ca2+ entry takes placethrough Ca2+ channels insensitive to these blockers or byalternative pathways such as the Na+/Ca2+ antiporter.

A Na+/Ca2+-exchange system exists in airway smoothmuscle (Chideckel et al. 1987). Supporting the notion thatthis mechanism is functional in the human airway smoothmuscle is our finding that bronchial muscle vigorouslycontracts when incubated in a Na+-free medium. Thesame phenomenon occurs in human, guinea-pig andbovine trachealis (Chideckel et al. 1987). The transientcontraction of human bronchus evoked by a Na+-free me-dium was abolished in Ca2+-free, EGTA (0.1 mM) PSS(present study). Therefore, the contraction induced by

Na+-free PSS results presumably from Ca2+ influx in ex-change for the efflux of Na+ down its concentration gradi-ent (Na+/Ca2+ antiporter acting in its reverse mode, i.e., asa Ca2+ influx pathway).

Since the Na+/Ca2+ antiporter appears functional in hu-man airway smooth muscle and incubation with Na+-freemedium suppressed the contractile response to ouabain(this study), the ouabain-induced contraction of humanisolated bronchus may be produced as a result of increased[Ca2+]i due to inhibition of basal Ca2+ efflux via Na+/Ca2+

exchange because of reduced [Na+]o/[Na+]i gradient or toCa2+ influx by reverse Na+/Ca2+ antiporter. A similar mech-anism operates for the contractile response to ouabain invascular smooth muscle (Iwamoto et al. 1992). These re-sults would be also consistent with the existence of a link-age between Na+/K+ pump and Na+/Ca2+ exchange in theplasmalemma of smooth muscle cells (Moore et al. 1993).

Amiloride produced a small transient contraction of hu-man isolated bronchus and markedly inhibited the subse-quently induced ouabain contraction (this study). This ef-fect may be related to the inhibitory action on the Na+/Ca2+ exchange reported for amiloride in vascular smoothmuscle (Bova et al. 1988), and represents additional ex-perimental evidence in favor of a role of the Na+/Ca2+ an-tiporter in ouabain contraction. However, amiloride is anon-selective agent and a number of alternative explana-tions for its inhibitory effect on ouabain-induced contrac-tion should be considered. Thus, amiloride is an inhibitorof Na+/H+ exchange at concentrations lower than those re-quired for Na+/Ca2+ exchange inhibition (Benos 1982);therefore, we investigated the contribution of this mecha-nism with a more selective amiloride derivative (see nextSection). Inhibition of the voltage-dependent Ca2+ chan-nel has been reported for amiloride in cardiac tissue butcontraction to ouabain is not related to Ca2+ entry throughvoltage-operated channels (see above). Amiloride has alsobeen reported to inhibit protein kinase C but selective in-hibitors of this enzyme failed to inhibit ouabain-inducedcontraction of human bronchus (see below).

Potential role of other plasmalemmal Na+ transportmechanisms in ouabain contraction

Airway smooth muscle has a functional Na+/H+ antiporterbut its role in airway smooth muscle responses to spasmo-gens has not been investigated in detail (Knox and Ajao1994). The amiloride derivative, DMA (30 µM), a con-centration sufficient to inhibit Na+/H+ antiport in airwaysmooth muscle (Rhoden et al. 2000), neither significantlyalter the resting tension of human isolated bronchus norsignificantly inhibit the ouabain-induced contraction. Thisfinding suggest that DMA-sensitive Na+/H+ exchange isnot relevant for ouabain contraction. However, the influ-ence of this mechanism cannot be definitely excluded sincethe Na+/H+ exchanger exists in multiple isoforms that dif-fer in their sensitivity to amiloride analogs without preciseinformation on the isoforms relevant in human airwaysmooth muscle (Rhoden et al. 2000).

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A Na+/K+/Cl- cotransporter has been described in air-way smooth muscle including human as an electricallyneutral, bidirectional pathway, driven by the transmembranegradients of all three ions but its role in the regulation ofairway smooth muscle contractility is yet unclear (Rhodenand Douglas 1995; Iwamoto et al. 2003). Bumetanide is aselective inhibitor of Na+/K+/Cl– cotransporter, and mea-surements of 86Rb+ influx in guinea pig trachealis demon-strate that ouabain and bumetanide act on distinct pathwaysof K+ uptake (Rhoden and Douglas 1995). In contrast, theouabain-induced 86Rb+ efflux from guinea pig trachealiswas abolished by bumetanide indicating that ouabainstimulates K+ efflux via the Na+/K+/Cl– cotransporter.However, bumetanide has relaxant or no effects on airwaysmooth muscle resting tension and does not alter direct re-sponses induced by different spasmogens yet studies onhuman airways are lacking (Iwamoto et al. 2001). Weshow that bumetanide relaxed human isolated bronchusbut had no significant effect on ouabain contraction. Theseresults suggest that the bumetanide-sensitive Na+/K+/Cl–

cotransporter does not appear to make a significant contri-bution to the spasmogenic effect of ouabain in human iso-lated bronchus.

Ouabain contraction is unrelated to protein kinase C activation

The activation of protein kinase C is involved in the toniccontraction of smooth muscle that can be maintained atlow levels of [Ca2+]i. A role of protein kinase C activationhas been demonstrated for the sustained second phase(tonic component) of the contractile response to histaminein human bronchus which is accompanied by low [Ca2+]ilevels; by contrast, the initial rapid contraction (phasiccomponent) subsequent to the immediate transient peak of[Ca2+]i was insensitive to protein kinase C inhibition (Yangand Black 1995). The contractile response to ouabain ismonophasic, slowly developing and well-sustained, and itwas not accompanied by a transient peak of [Ca2+]i simi-lar to that observed after histamine but by relatively lowand sustained [Ca2+]i levels along the progressive built ofthe tonic contraction. However, the ouabain-induced con-traction was resistant to staurosporine and calphostin C,two selective inhibitors of protein kinase C, and therefore,the activation of this kinase is not apparently involved inthe tonic contraction to ouabain in human bronchus. Thisresult, taken together with the lack of accumulation ofinositol phosphates (see above), indicates that ouabain isnot activating any of the different phospholipase C iso-forms present in airway smooth muscle (Chilvers et al.1994).

Ouabain impaired relaxant but not contractile responsesof human isolated bronchus

In human bronchial preparations, ouabain (10 µM) pro-duces a sustained contraction and therefore the influence

of ouabain in the responses to spasmogens was exploredfrom a raised baseline tone. Under these conditions, cu-mulative addition of ACh and histamine produced con-centration-response curves that did not significantly differin their maximal effects and in their potency (–log EC50)values from those obtained in control untreated tissues.These findings are consistent with other data in this studyshowing that ouabain (10 µM) did not alter the inositolphosphate accumulation produced by ACh or the [Ca2+]ichange in response to histamine. In addition, the transientcontraction produced by ryanodine was not modified inouabain-treated tissues (this study). These findings extendthe observation by Knox et al. (1990) that ouabain (10 µM)did not alter methacholine responsiveness and sensitivityin human bronchial rings. Black et al. (1984) reported thatouabain markedly depressed the concentration-effect curvesto histamine, KCl and carbachol in human isolated bronchusbut they used a higher concentration (50 µM) and data onresponsiveness and sensitivity of preparations or the con-centration-response curves were not provided in theirstudy.

By contrast with the lack of effect of ouabain on spas-mogenic responses, the relaxation to a number of drugsacting through different mechanisms were depressed byouabain. Thus, ouabain reduced the relaxation producedby activation of β-adrenoceptors with isoprenaline, acti-vation of adenylyl cyclase with forskolin, opening of KATPchannels with levcromakalim, and activation of guanylylcyclase with sodium nitroprusside. These results extend tothe human bronchus previous findings of inhibition byouabain of relaxations produced by isoprenaline, salbuta-mol, prostaglandin E2, forskolin, vasoactive intestinal pep-tide, and sodium nitroprusside in canine, guinea-pig andrabbit trachealis (Morrison and Vanhoutte 1996). Althoughdirect biochemical evidence of stimulation of Na+/K+-AT-Pase activity by the relaxants tested in human bronchus isnot provided in this work, others have demonstrated inguinea-pig trachea that vasoactive intestinal peptide effec-tively increases ouabain-sensitive uptake of 86Rb as a in-dex of Na+/K+-ATPase activity (Morrison and Vanhoutte1996). In the case of cyclic AMP-dependent relaxants, en-zyme activation may be accomplished by direct phosphor-ylation of Na+/K+-ATPase α-subunits by protein kinase Abut the mechanism underlying activation by other relax-ants is not yet established.

In conclusion, ouabain produces depolarization and asustained contraction of human airway smooth muscle.Contraction results from extracellular Ca2+ entry with par-ticipation of a Na+/Ca2+ exchange mechanism but no in-volvement of voltage-operated channels. Although ex-trapolation of in vitro data to the in vivo situation is un-certain, our data suggest that the airway hyperreactivity tohistamine found in allergic patients after inhaled ouabainis not likely to be related with an enhancement of respon-siveness and sensitivity to spasmogens but to impaired re-laxant mechanisms.

Acknowledgements The present work was supported in part bygrants SAF2000–0144 and SAF2002–04667 from CICYT (Min-istry of Science and Technology, Spain). The authors are indebted

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to the teams of the Services of Thoracic Surgery and Pathology ofthe Hospital La Fe and Hospital Clínico of the University of Va-lencia for making the human lung tissue available to us. We alsothank V. Villagrasa, P. Santamaría and S. Martí for help with ex-periments.

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