Clinical Science ( 1990) 79,325-330
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Effect of sodium-transport inhibitors on bronchial reactivity in vivo ALAN J. KNOX, JOHN R. BRITTON
AND
ANNE E. TATTERSFIELD
Respiratory Medicine Unit, City Hospital, Nottingharn, U.K.
(Received 29 June 1989/18 April 1990; accepted 22 May 1990)
SUMMARY
1. We have recently shown that ouabain, an inhibitor of Na+/K+-adenosine triphosphatase, causes contraction of bovine and human airways in vitro, and that amiloride causes relaxation and inhibits receptor-operated contraction in bovine trachealis. 2. To determine whether such drugs alter bronchial reactivity it7 vivo, we have studied the effect of oral digoxin (an inhibitor of Na+/K+-adenosine triphosphatase) and oral and inhaled amiloride on bronchial reactivity to histamine in three double-blind, placebocontrolled studies. 3. Histamine reactivity was measured as the provocative dose causing a 20% reduction in the forced expiratory volume in 1 s (PD,,,FEV,) or, when normal subjects were included, the provocative dose causing a 35% reduction in the specific airways conductance (PD,,sGaw); the results are given as geometric mean values. 4. In study 1, 13 atopic asthmatic subjects were b’ wen 20 mg of oral amiloride or placebo on separate days. Two hours after the drug, the geometric mean PD,,,FEV, for histamine was 0.43 pmol after amiloride and 0.54 pmol after placebo (95% confidence intervals for the difference: 0.9 to - 0.2 doubling doses of histamine; f’= 0.2). 5. In study 2, six normal and 24 atopic asthmatic men inhaled 10 ml of l o - ? mol/l amiloride or diluent control in a crossover study. The mean values of PD,,sCaw for histamine immediatelv after inhalation of amiloride and placebo were 3.0 pmol and 4.3 pmol, respectively, in the normal subjects (95% confidence intervals for the difference: -0.53 to 1.52 doubling doses, P=0.2), and 0.33 pmol and 0.29 pmol in the asthmatic subjects (95% confidence intervals for the difference: - 0.95 to 0.57 doubling doses; f’= 0.6). 6. In study 3,24 atopic asthmatic men were treated for 7 days with placebo or oral digoxin (1.5 mg loading dose Correspondence: Dr Alan J. Knox, Respiratory Medicine Unit, City Hospital, Nottingharn NG5 1 PB, U.K.
plus 0.25 mg twice daily for 6 days). The PD,,,FEV, for histamine was measured before, 12 h after the loading dose and on day 7 of treatment. The change in PD2,,FEV, did not differ significantly after digoxin and placebo, after either 1 day’s treatment [mean (95% confidence intervals) difference: 0.56 doubling dose ( - 0.37 to 1.5 doubling dose)] or 7 day’s treatment [mean (95% confidence intervals) difference: 0.3 doubling dose ( - 1.23 to 1.8 doubling doses)]. 7. Although our work it7 vitro has suggested that membrane sodium transport may play an important role in determining airway smooth muscle contractility, we have been unable to demonstrate any effect of the sodiumtransport inhibitors amiloride and digoxin on histamine reactivity in these studies. Key words: amiloride, bronchial reactivity, digoxin, histamine.
Abbreviations: DD, doubling dose; FEV,, forced expiratory volume in 1 s; Na+/K+-ATPase, Na+/K+-adenosine triphosphatase; PD,,,FEV,, provocative dose causing a 20% reduction in the forced expiratory volume in 1 s; PD,,sGaw, provocative dose causing a 35% reduction in the specific airways conductance; sGaw, specific airways conductance. INTRODUCTION
Recent evidence suggests that a high salt diet increases bronchial reactivity and may influence asthma mortality [ 1-41, but the underlying mechanism is unknown. One explanation for this association would be an increase in intracellular sodium in airway smooth muscle after salt ingestion. An increase in intracellular sodium would be expected to cause an increase in intracellular calcium, via Na+/Ca’+ exchange, which would alter contractility. An increase in intracellular sodium could occur directly, as despite all the homoeostatic mechanisms which operate to minimize changes in extra-
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cellular sodium, small changes in plasma sodium are still apparent after several days on a high salt diet [5].Much more likely, however, an increase in intracellular sodium could be produced by the circulating inhibitors of Na+/ K+-adenosine triphosphatase (Na+/K+-ATPase) as described by dewardener et al. [6] in subjects taking a high salt diet, the increase in the concentration of these inhibitors probably occurring as a result of extracellular volume expansion. In a series of studies in vitro we have shown that the sodium-transport inhibitors ouabain and amiloride alter the contractile properties of bovine and human airway smooth muscle. Ouabain, an inhibitor of Na+/K+-ATPase, contracted the airway smooth muscle [7], as shown by previous workers [8, 91. In contrast, amiloride, an inhibitor of Na+/ H + exchange, sodium entry and possibly Na+/Ca2+ channels, inhibited the contractile response to histamine and carbachol[7]. The airway response to a constrictor agent in vivo will depend on the net response of various cells in the airways in addition to airway smooth muscle, epithelial cells, inflammatory cells, vascular endothelium and neurons. Raeburn et al. [lo] have also suggested that N a + / H + exchange and Na+/K+-ATPase may regulate release of epithelial relaxant factor [lo], and several studies have suggested that sodium-transport activity regulates inflammatory cell function [ 11-14]. Thus alterations in tissue sodium-transport systems could modify asthma by effects on inflammatory cells, airway smooth muscle or epithelium. Studies in guinea pigs in vivo have shown that ouabain is a bronchoconstrictor and increases histamine reactivity [15], and that amiloride attenuates the airway response to inhaled allergen [ 161. We have therefore performed a series of studies in normal and asthmatic subjects to determine whether inhibition of Na+/K+-ATPase or amiloride sensitive sodium transport alters bronchial reactivity to histamine. We performed three separate studies looking at the effect of oral amiloride, inhaled amiloride and oral digoxin.
MATERIALS AND METHODS Subjects The asthmatic subjects were recruited from the City Hospital Asthma Register. All were atopic, had a forced expiratory volume in 1 s (FEV,) > 70% of predicted, had
previously demonstrated an increase in FEV, of 20"/0 or more after 200 pg of inhaled salbutamol, had a provocative dose of histimine causing a 20% reduction in FEV, (PD2,,FEV,)of < 4 pmol and were clinically stable. All were controlled on inhaled therapy alone, and all were taking inhaled P-adrenoceptor agonists. Details of the number of patients in each study and of their additional treatment are given in Table 1. The normal subjects were all fit, healthy male volunteers, aged 18-45 years, with no previous history of allergic diseases. None of the subjects had a history of cardiac disease or hypertension and all were non-smokers with no history of upper respiratory tract infection in the preceeding 6 weeks. Written consent was obtained from each subject; the study design was approved by the City Hospital Ethics Committee.
Methods FEV, was measured on a dry bellows spirometer (Vitalograph) taking the higher of two measurements within 100 ml. All measurements were made with the subjects seated. Specific airways conductance (sCaw) measurements were made in a whole-body plethysmograph (Gould 2800 Autobox), attached to a computer, each value being a mean of nine separate measurements. Serum digoxin was measured by fluorescent polarization immunoassay (Abbot) which had an intra-assay coefficient of variation of 4.7-6.75%.
Bronchial reactivity If the subjects had no previous experience of challenge test procedures they had one preliminary visit to familiarize them with the procedure. Histamine challenge was performed by a method based on that of Yan et al. [17] using De Vilbiss no. 40 hand-held nebulizers with an output ranging from 0.0025 to 0.0035 ml/activation. After baseline measurement of FEV,, subjects inhaled three puffs of 0.9% (w/v) NaCl (saline) followed by doubling doses (DD) of histamine acid phosphate (BDH Chemicals, Poole, Dorset, U.K.) over the dose range 0.03-32 pmol. In studies 1 and 3 the response was measured as FEV,, 1 min after each inhalation. The test was terminated when FEV, had fallen by 20% from post-saline values. In study 2, in which normal subjects participated, the response was measured as sCaw, 1 min after each
Table 1. Details of subjects and treatment in the three studies Study 1: oral
Study 2 : inhaled
oral
amiloride
amiloride
digoxin
Type of subjects
13 asthmatic
24 asthmatic
Sex
Mixed 2 1-68
6 normal, 24 asthmatic Male 18-45 All 8 2
All 14 1
Age range (years) No. taking
Inhaled P-agonists Inhaled steroids Inhaled cromoglvcate
All
4 4
Study 3:
Male
18-45
Sodium transport and bronchial reactivity inhalation, and the test was terminated when s c a w had fallen by 35% from post-saline values. The provocative dose of histamine causing a 20% fall in FEV, (PD2,,FEV,) or a 35% fall in sCaw (PD,,sGaw) was calculated by interpolation on log dose-response plots. Protocols All three studies were double-blind and placebocontrolled. Subjects were instructed to continue their usual treatment throughout the study periods, but to avoid using /3-adrenergic agonists for 6 h before each laboratory visit. All visits for each subject were at the same time of day. Study 1: effect of oral amiloride on bronchial reactivity to histamine. Thirteen asthmatic subjects, aged 2 1-68 years, were studied on 2 days in the same week, ingesting 20 mg of oral amiloride (Morson Pharmaceuticals, Hoddesden, Herts, U.K.) or placebo in randomized crossover fashion. Measurement of FEV, and histamine reactivity (PD,,,FEV, ) was made 2 h after drug ingestion. Study 2: effect of inhaled amiloride on bronchial reactivity to histamine. We studied six normal and 24 asthmatic men, aged 18-45 years. Subjects attended on 2 days within the same week. After baseline measurement of sGaw, subjects received either inhaled amiloride or control diluent in randomized crossover fashion. Drugs were administered via an Inspiron Mini-neb nebulizer (Bard, Sunderland, U.K.) driven by air at 8 I/min. Ten millimetres of 10- mol/l amiloride hydrochloride (Sigma, Poole, Dorset, U.K.) or 10 ml of distilled water (control solution) was placed in the nebulizer chamber and nebulized to dryness. Repeat s Caw measurements were made immediately after inhalation and were followed by a histamine challenge test to measure PD,,sGaw. Study 3: effect of oral digoxin on FEV,, PDz0FEV, for histamine and home peak flow rate. We studied 24 asthmatic men, aged 18-45 years. Subjects attended the laboratory on day 1 for initial assessment and measurement of FEV, and histamine reactivity. They were given a mini-Wright peak flow meter and asked to record twice daily peak flow rate (best of three) for the next 2 weeks. On day 8 subjects returned for a repeat measurement of FEV, and histamine reactivity (PD,,FEV,). They were then randomized to receive either oral digoxin (Wellcome, Crewe, U.K.) 0.5 mg three times daily for 1 day followed by 0.25 mg twice daily for 6 days or matched placebo for 7 days. FEV, ,histamine reactivity and serum digoxin level were measured on day 9 and day 15 (i.e. after 1 and 7 days of digoxin or placebo treatment).
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after the drug between amiloride and placebo in study 2 were compared by using Student's paired t-test. In study 3, changes in FEV, and PD2,,FEVI after digoxin and placebo were compared by analysis of variance. Mean peak flow for weeks 1 and 2 were calculated for each subject and the change in peak flow (week 2 minus week 1) after digoxin and placebo was compared by analysis of variance. P< 0.05 was regarded as significant. All three studies had 95% power to detect differences of one DD of histamine. RESULTS Study 1: effect of oral amiloride on FEV, and bronchial reactivity to histamine The mean (SD) FEV, was 2.8 (0.9) litres and 2.8 (0.7) litres after amiloride and placebo, respectively. The geometric mean (95% confidence intervals) PD2,,FEVI was 0.43 (0.18-1.05) pmol and 0.54 (0.22-1.37) pmol after amiloride and placebo, respectively (Fig. 1). The mean (95% confidence intervals) difference in PD,,,FEV, between amiloride and placebo was 0.34 DD (-0.2 to + 0.9 DD) of histamine ( P = 0.2). Study 2: effect of inhaled amiloride on bronchial reactivity to histamine There was no difference between the placebo and amiloride study days for the mean baseline sCaw, the mean sCaw immediately after inhaled treatment or the change in sCaw after treatment in either the six normal or the 24 asthmatic subjects (Fig. 2). The geometric mean (95% confidence intervals) PD,,sCaw values after amiloride and placebo were 3.0 (1.6-5.7) pmol and 4.3 (2.3-7.9) pmol in the normal subjects, and 0.33 (0.17-0.64) pmol and 0.29 (0.17-0.50) pmol, respectively, in the asthmatic subjects (Fig. 3). The mean (95% confidence intervals) difference in PD,,sGaw between amiloride and placebo was 0.5 ( - 0.53 to + 1.52) DD in the normal subjects ( P = 0.2) and - 0.2 ( - 0.94 to + 0.56) DD in the asthmatic subjects ( P = 0.6).
'"1
Analyses Log-transformed provocative dose values were used in all analyses. For studies 1 and 2, PD2,,FEV, and PD,,sCaw values after drug and placebo were compared by using Student's paired t-test. We expressed the change in PD,,FEV, and PD,,sCaw in terms of DD of histamine. Differences in baseline sGaw and the change in sGaw
0.1
'
I
I
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Amiloride
Fig. 1. Geometric m e a n k s ~PD2,,FEV, ~ 2 h after oral amiloride or placebo in 13 subjects with asthma.
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1.8 1.6
I.% 1.2 "
I
1
I
I
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Fig. 2. Effect of inhaled amiloride ( 0 )or placebo ( 0 )on sCaw in six normal subjects ( u ) and 24 asthmatic subjects ( b ) Values . are means fSEM. Study 3: effect of oral digoxin on FEV,, PD,,FEV, for histamine and home peak flow rate
There was no significant difference between digoxin and placebo in the change in FEV, values after drug (day 8 versus days 9 and 1 S ) , in the change in home peak flow rate after the drug or in the change in PD,,,FEV, after the drug (Figs. 4a and 4b). The geometric mean PDI,,FEV, values on days I , 8, 9 and 15 were 0.7, 0.57, 0.51 and 0.63 pmol o n digoxin, and 0.81, 0.74, 0.89 and 0.86 pmol on placebo (Fig. 4c). The mean (95% confidence intervals) difference in PDI,,FEV, between digoxin and placebo was 0.56 ( - 0.37 to 1S ) DD of histamine after 1 days' treatmcnt ( P = 0 . 2 ) and 0.3 ( - 1.23 to 1.8) DD after 7 days' treatment (1'=0.6). The mean (range) serum digoxin level was 1.1 (0.6-2.5) pg/l after 1 day's treatment and 1.2 (0.6-2.7) pg/l after 7 day's treatment. DISCUSSION
The aim of the present study was to determine whether t w o inhibitors of sodium transport that have been shown to alter airway smooth muscle contractility irz vitro would alter bronchial reactivity in patients with asthma. We studied amiloride which is known to block several sodium transporters including sodium-entry channels found in epithelial tissue, and the Na'/H+ exchanger which is thought to be present in all cells [ 181. Amiloride has also been shown to inhibit Na+/Ca?+ exchange, at lcast in cardiac tissue 119, 201. In studies in bovine trachea irz vitro, we have shown that amiloride inhibits the contractile response to both histamine and carbachol in doses of 10 pmol/l or greater [ 7 ] .Souhrada et al. 1161 have previously shown that in the guinea-pig amiloride attenuates the airway response to antigen in vivo. In study 1, we used the highest recommended dose of oral amiloride but were unable to show any alteration in histamine responsiveness. The diuretic action of amiloride is due to inhibition of epithelial sodium-entry channels and these are blocked by approximately 1 pmol/l concentrations of amiloride [ 181. The plasma concentrations obtained during oral therapy with 5-20 mg of amiloride are slightly greater
.,. .
1
I
Placebo
Amiloride
Fig. 3. Geometric mean fSEM PD,,sGaw after inhaled amiloride or placebo in six normal subjects ( 0 )and 24 asthmatic subjects ( 0 ) .
than 1 pmol/l 1181 and should therefore have blocked sodium-entry channels in airway epithelium in our study. Local drug concentrations in airway smooth muscle may have been lower than those required to inhibit smooth muscle contraction in vitro, however. In study 2, we therefore tried to obtain the maximum possible local concentrations of drug by giving amiloride by inhalation to the limit of its solubility in water ( l o - ' mol/l). The resulting hypo-osmolarity may account for the small, although nonsignificant, reduction in sGaw on both placebo and active treatment days in asthmatic subjects (Fig. 2). The reason we did not see more bronchoconstriction may be due to the fact that we used jet rather than ultrasonic nebulizers 1211. We studied only young atopic men, as the relationship between dietary salt intake and histamine reactivity is stronger in this group of subjects 111, and used PD,,sGaw as a measure of reactivity. since it is more sensitive than PD2,,FEV,and would therefore be more likely to detect small changes 122).Despite this, we again saw no effect of amiloride on histamine responsiveness or sGaw. We have considered whether we achieved adequate tissue concentrations of amiloride. Waltner et ul. [23]have looked at the deposition of inhaled amiloride in normal subjects and patients with cystic fibrosis and showed that a concentration of 10-4 mol/l in airway surface liquid could be achieved by nebulizing 5 x l o - , mol/l amiloride. As we used a concentration of lo-' mol/l, the concentration we achieved in airway surface liquid should have been mol/l. How much of this would approximately 2 x then be absorbed across the bronchial mucosa is uncertain, but it is possible that we were still unable to obtain levels in bronchial smooth muscle of the magnitude required to produce an effect in vitro. Work in sheep suggests that amiloride is taken up into the bloodstream very rapidly after being inhaled, which would further reduce tissue concentrations 1241. It is also possible that an effect on airway smooth muscle was masked by an opposing effect on airway epithelium or inflammatory cells. This would seem unlikely as inhibition of Na' /H ' exchange should reduce inflammatory cell activity [ 12-14] and increase the release of epithelial relaxant
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responsiveness [7].This contrasts with a study by Agrawal & Hyatt [ 151 in guinea pigs in vivo, where ouabain caused bronchoconstriction and increased histamine responsiveness. We have previously shown no change in histamine responsiveness in asthmatic subjects after inhaled ouabain [25], but ouabain may not penetrate bronchial mucosa well [26]. We therefore gave oral digoxin in the present study, as it is well absorbed orally and serum concentrations can be measured easily [27]. The serum concentrations we achieved have been shown to produce widespread reduction in tissue Na+/K+-ATPase activity in vivo [28] and it seems likely, therefore, that this occurred in airway smooth muscle in our study. Despite this we did not see any bronchoconstriction or alteration in bronchial reactivity. The reasons for the disparity between these findings and our findings in vitro are not obvious. Digoxin has effects other than Na+/K+-ATPase inhibition, including parasympathomimetic activity, but this would be expected to increase bronchoconstriction. It can also potentiate the effects of circulating catecholamines [27] and this could mask a bronchoconstrictor effect. It is also possible that the Na+/K+-ATPaseinhibition produced by digoxin in vivo was less than that produced by ouabain in vitro. Our studies show that although alterations in cell membrane sodium transport can alter the contractile state of bovine and human airway smooth muscle in vitro, the sodium-transport inhibitors currently available, in the doses we are able to give, d o not alter bronchomotor tone or bronchial reactivity in vivo. Amiloride analogues with greater activity against Na+/H+ exchange and Na+/Ca? exchange than the parent compound have recently been synthesized and might be more effective [29,30]. +
Time (days)
ACKNOWLEDGMENTS We thank the Asthma Research Council for financial support, Dr C. Marenah for measuring serum digoxin levels, and Mr A. Wisniewski and Mrs S. Cooper for technical assistance. REFERENCES
.
0 0
2
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14
Fig. 4. Mean k SEM home peak flow rate (PEF ) for weeks 1 and 2 (a), m e a n k ~FEV, ~ ~ (6) and geometric mean k SEM PD,,,FEV, (c) in 24 asthmatic patients before (days 1-7) and after (days 8-15) receiving digoxin ( 0 )or placebo (0).
factor [lo]. Both of these effects should potentiate any relaxant effect of amiloride. In study 3 we studied the effect of digoxin, a Na+/K'ATPase inhibitor, on histamine reactivity. In our studies in vitro, the Na' /K'-ATPase inhibitor ouabain caused contraction of bovine trachea but did not alter histamine
1. Burney, P.G.J. A diet rich in salt may potentiate asthma: epidemiological evidence for a new hypothesis. Chest 1987; 91,183s-6s. 2. Burney, P.G.J., Britton, J.R. & Chinn, S. et al. Response to inhaled histamine and 24 hour sodium excretion. Br. Med. J. 1986; 292, 1486-6. 3. Javaid, A., Cushley, M.J. & Bone, M.F. Effect of dietary salt on bronchial reactivity to histamine in asthma. Br. Med. J. 1988; 297,454. 4. Burney, P.G.J., Neild, J., Twort, C. et al. The effects of changing dietary sodium on the bronchial response to histamine. Thorax 1989: 44,36-4 I . 5. Sagnella, G.A., Markandu, N.D., Shore, A.C., Forsling, M.L. & MacGregor, G.A. Plasma atrial natriuretic peptide: its relationship to changes in sodium intake, plasma renin activity and aldosterone in man. Clin. Sci. 1987; 7 2 , 2 5 3 0 . 6. De Wardener, H.E., MacGregor, G.A., Clarkson, E.M., Alaghband-Zadeh, J., Bitensky, L. & Chayen, J. Effect of sodium intake on the ability of human plasma to inhibit renal adenosine triphosphatase in vitro. Lancet 198 1 ; i, 41 1-12.
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7. b o x , A.J., Ajao, P., Britton, J.R. & Tattersfield, A.E. Effect of sodium transport inhibitors on airway smooth muscle contractility in vitro. Clin. Sci. 1990: 7 9 , 3 15-23. 8. Chideckel, E.W., Frost, J.L., Mike, P. & Fedan, J. S. The effect of ouabain on tension in isolated respiratory tract smooth muscle of humans and other species. Br. J. PharmaCOI. 1987; 92,609-14. 9. Bullock, C.G., Fettes, J.J.F. & Kirkpatrick, C.T. Tracheal smooth muscle - second thoughts on sodium-calcium exchange. J. Phyisol. (London) 1981; 318,46P. 10. Raeburn, D., Farmer, S.G., Hay, D.W.P. & Fedan, S. Airway epithelium (EPI) modulates Na'/K+ pumping and N a + / H + exchange in guinea pig trachealis (GPT). Fed. Proc. Fed. Am. SOC.Exp. Biol. 1987; 46, 376. 11. Elliot, G.R.. Braquet. P.. Batenburg, M. & Bonta, 1.1. Changes in resident rat peritoneal macrophage eicosanoid release induced by altering the buffer K+/Na+ ratio are calcium dependent. Biochim. Biophys. Acta 1985; 837, 213-21. 12. Grinstein, S. & Furuya, W. Characterisation of the amiloride sensitive Na'-H' antiport o f human neutrophils. Am. J. Physiol. 1986; 250, C283-9 I . 13. Simchowitz, L. Chemotactic factor induced activation of Na+/H+ exchange in human neutrophils. J. Biol. Chem. 1985; 260, 13237-47. 14. Nasmith, P.E. & Grinstein, S. Impairment of Na'/H' exchange underlies inhibitory effects of Na' free medium on leukocyte function. FEBS Lett. lY86; 202, 79-85. 15. Agrawal, K.P. & Hyatt, R.E. Airway response to inhaled ouabain and histamine in conscious guinea pigs. J. Appl. Physiol. 1986; 60, 2089-93. 16. Souhrada, M., Souhrada. M.H. & Souhrada, J.F. The inhibition o f sodium intlux attenuates airway response to a specific antigen challenge. Br. J. Pharmacol. 1988; 93, 884-92. 17. Yan, K., Salome, C . & Woolcock, A.J. Rapid method for measurement o f bronchial responsiveness. Thorax 1983; 38, 760-5. 18. Benos. D.J. Amiloride: a molecular probe of sodium transport in tissues and cells. Am. J. Physiol. 1982; 242, CI 3 1-45.
19. Floriani, M. & Luciani, S. Amiloride: relationship between cardiac effects and inhibition of Na+/Ca?+exchange. Eur. J. Pharmacol. 1984; 105,3 17-22. 20. Debetto, P., Floriani, M., Carpanedo, F. & Luciani, S. Inhibition of the Na+/Caz+ exchange in cardiac vesicles by amiloride. Life Sci. 1987; 40, 1523-30. 21. Smith, C.M. & Anderson, S.D. Inhalation provocation tests using nonisotonic aerosols. J. Allergy Clin. Immunol. 1989; 84,781-90. 22. Dehaut, P., Rachiele, A,, Martin, R.R. & Malo, J.L. Histamine dose response curves in asthma: reproducibility and sensitivity of different indices to assess response. Thorax 1983; 3 8 , s 16-22. 23. Waltner, W.E., Church, N.L.. Gatzy, J.T., Boucher, R.C. & Knowles, M.R. Deposition, pharmacokinetics, and toxicity of amiloride aerosol in normal and cystic fibrosis subjects. Am. Rev. Respir. Dis. 1987; 135, A288. 24. Mentz, W.M., Brown, J.B., Friedman, M., Stutts, M.J., Gatzy, J.T. & Boucher, R.C. Deposition, clearance and effects of inhaled amiloride in sheep airways. Am. Rev. Respir. Dis. 1986; 134,938-43. 25. Knox, A.J., Tattersfield, A.E. & Britton, J.R. The effect of inhaled ouabain on bronchial reactivity to histamine in man. Br. J. Clin. Pharmacol. 1988; 25, 758-60. 26. Widdicombe, J.H., Ueki, I.F.. Bruderman, I. & Nadel, J.A. The effects of sodium substitution and ouabain on ion transport by dog tracheal epithelium. Am. Rev. Respir. Dis. 1979; 120.385-92. 27. Smith. I.W. Digitalis. Mechanisms of action and use. N. Engl. J. Med. 1988; 318, 358-65. 28. Boon, N., Pugh, S.E., Hallis, K.F., Aronson, J.K. & Grahame-Smith, D.G. In vivo cation transport during short term and long term digoxin therapy. Br. J.'Clin. Pharmacol. 1986; 22. 27-30. 29. Siegl, P.K.S., Cragoe, E.J., Trumble, M.J. & Kaczorowski, G.J. Inhibition of Na'/Ca'' exchange in membrane vesicles and papillary muscle preparations by analogues of amiloride. Proc. Nat. Acad. Sci. U.S.A. 19x4; 81. 3238-42. 30. Vigne, P., Frelin, C.. Cragoe. E.J. & Ladzunski, M. Structure-activity relationships o f amiloride and certain of its analogues in relation to blockage o f the Na+/H' exchange system. Mol. Pharmacol. 1983; 25, I3 1-6.
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Effect of sodium-transport inhibitors on bronchial reactivity in vivo ALAN J. KNOX, JOHN R. BRITTON
AND
ANNE E. TATTERSFIELD
Respiratory Medicine Unit, City Hospital, Nottingharn, U.K.
(Received 29 June 1989/18 April 1990; accepted 22 May 1990)
SUMMARY
1. We have recently shown that ouabain, an inhibitor of Na+/K+-adenosine triphosphatase, causes contraction of bovine and human airways in vitro, and that amiloride causes relaxation and inhibits receptor-operated contraction in bovine trachealis. 2. To determine whether such drugs alter bronchial reactivity it7 vivo, we have studied the effect of oral digoxin (an inhibitor of Na+/K+-adenosine triphosphatase) and oral and inhaled amiloride on bronchial reactivity to histamine in three double-blind, placebocontrolled studies. 3. Histamine reactivity was measured as the provocative dose causing a 20% reduction in the forced expiratory volume in 1 s (PD,,,FEV,) or, when normal subjects were included, the provocative dose causing a 35% reduction in the specific airways conductance (PD,,sGaw); the results are given as geometric mean values. 4. In study 1, 13 atopic asthmatic subjects were b’ wen 20 mg of oral amiloride or placebo on separate days. Two hours after the drug, the geometric mean PD,,,FEV, for histamine was 0.43 pmol after amiloride and 0.54 pmol after placebo (95% confidence intervals for the difference: 0.9 to - 0.2 doubling doses of histamine; f’= 0.2). 5. In study 2, six normal and 24 atopic asthmatic men inhaled 10 ml of l o - ? mol/l amiloride or diluent control in a crossover study. The mean values of PD,,sCaw for histamine immediatelv after inhalation of amiloride and placebo were 3.0 pmol and 4.3 pmol, respectively, in the normal subjects (95% confidence intervals for the difference: -0.53 to 1.52 doubling doses, P=0.2), and 0.33 pmol and 0.29 pmol in the asthmatic subjects (95% confidence intervals for the difference: - 0.95 to 0.57 doubling doses; f’= 0.6). 6. In study 3,24 atopic asthmatic men were treated for 7 days with placebo or oral digoxin (1.5 mg loading dose Correspondence: Dr Alan J. Knox, Respiratory Medicine Unit, City Hospital, Nottingharn NG5 1 PB, U.K.
plus 0.25 mg twice daily for 6 days). The PD,,,FEV, for histamine was measured before, 12 h after the loading dose and on day 7 of treatment. The change in PD2,,FEV, did not differ significantly after digoxin and placebo, after either 1 day’s treatment [mean (95% confidence intervals) difference: 0.56 doubling dose ( - 0.37 to 1.5 doubling dose)] or 7 day’s treatment [mean (95% confidence intervals) difference: 0.3 doubling dose ( - 1.23 to 1.8 doubling doses)]. 7. Although our work it7 vitro has suggested that membrane sodium transport may play an important role in determining airway smooth muscle contractility, we have been unable to demonstrate any effect of the sodiumtransport inhibitors amiloride and digoxin on histamine reactivity in these studies. Key words: amiloride, bronchial reactivity, digoxin, histamine.
Abbreviations: DD, doubling dose; FEV,, forced expiratory volume in 1 s; Na+/K+-ATPase, Na+/K+-adenosine triphosphatase; PD,,,FEV,, provocative dose causing a 20% reduction in the forced expiratory volume in 1 s; PD,,sGaw, provocative dose causing a 35% reduction in the specific airways conductance; sGaw, specific airways conductance. INTRODUCTION
Recent evidence suggests that a high salt diet increases bronchial reactivity and may influence asthma mortality [ 1-41, but the underlying mechanism is unknown. One explanation for this association would be an increase in intracellular sodium in airway smooth muscle after salt ingestion. An increase in intracellular sodium would be expected to cause an increase in intracellular calcium, via Na+/Ca’+ exchange, which would alter contractility. An increase in intracellular sodium could occur directly, as despite all the homoeostatic mechanisms which operate to minimize changes in extra-
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cellular sodium, small changes in plasma sodium are still apparent after several days on a high salt diet [5].Much more likely, however, an increase in intracellular sodium could be produced by the circulating inhibitors of Na+/ K+-adenosine triphosphatase (Na+/K+-ATPase) as described by dewardener et al. [6] in subjects taking a high salt diet, the increase in the concentration of these inhibitors probably occurring as a result of extracellular volume expansion. In a series of studies in vitro we have shown that the sodium-transport inhibitors ouabain and amiloride alter the contractile properties of bovine and human airway smooth muscle. Ouabain, an inhibitor of Na+/K+-ATPase, contracted the airway smooth muscle [7], as shown by previous workers [8, 91. In contrast, amiloride, an inhibitor of Na+/ H + exchange, sodium entry and possibly Na+/Ca2+ channels, inhibited the contractile response to histamine and carbachol[7]. The airway response to a constrictor agent in vivo will depend on the net response of various cells in the airways in addition to airway smooth muscle, epithelial cells, inflammatory cells, vascular endothelium and neurons. Raeburn et al. [lo] have also suggested that N a + / H + exchange and Na+/K+-ATPase may regulate release of epithelial relaxant factor [lo], and several studies have suggested that sodium-transport activity regulates inflammatory cell function [ 11-14]. Thus alterations in tissue sodium-transport systems could modify asthma by effects on inflammatory cells, airway smooth muscle or epithelium. Studies in guinea pigs in vivo have shown that ouabain is a bronchoconstrictor and increases histamine reactivity [15], and that amiloride attenuates the airway response to inhaled allergen [ 161. We have therefore performed a series of studies in normal and asthmatic subjects to determine whether inhibition of Na+/K+-ATPase or amiloride sensitive sodium transport alters bronchial reactivity to histamine. We performed three separate studies looking at the effect of oral amiloride, inhaled amiloride and oral digoxin.
MATERIALS AND METHODS Subjects The asthmatic subjects were recruited from the City Hospital Asthma Register. All were atopic, had a forced expiratory volume in 1 s (FEV,) > 70% of predicted, had
previously demonstrated an increase in FEV, of 20"/0 or more after 200 pg of inhaled salbutamol, had a provocative dose of histimine causing a 20% reduction in FEV, (PD2,,FEV,)of < 4 pmol and were clinically stable. All were controlled on inhaled therapy alone, and all were taking inhaled P-adrenoceptor agonists. Details of the number of patients in each study and of their additional treatment are given in Table 1. The normal subjects were all fit, healthy male volunteers, aged 18-45 years, with no previous history of allergic diseases. None of the subjects had a history of cardiac disease or hypertension and all were non-smokers with no history of upper respiratory tract infection in the preceeding 6 weeks. Written consent was obtained from each subject; the study design was approved by the City Hospital Ethics Committee.
Methods FEV, was measured on a dry bellows spirometer (Vitalograph) taking the higher of two measurements within 100 ml. All measurements were made with the subjects seated. Specific airways conductance (sCaw) measurements were made in a whole-body plethysmograph (Gould 2800 Autobox), attached to a computer, each value being a mean of nine separate measurements. Serum digoxin was measured by fluorescent polarization immunoassay (Abbot) which had an intra-assay coefficient of variation of 4.7-6.75%.
Bronchial reactivity If the subjects had no previous experience of challenge test procedures they had one preliminary visit to familiarize them with the procedure. Histamine challenge was performed by a method based on that of Yan et al. [17] using De Vilbiss no. 40 hand-held nebulizers with an output ranging from 0.0025 to 0.0035 ml/activation. After baseline measurement of FEV,, subjects inhaled three puffs of 0.9% (w/v) NaCl (saline) followed by doubling doses (DD) of histamine acid phosphate (BDH Chemicals, Poole, Dorset, U.K.) over the dose range 0.03-32 pmol. In studies 1 and 3 the response was measured as FEV,, 1 min after each inhalation. The test was terminated when FEV, had fallen by 20% from post-saline values. In study 2, in which normal subjects participated, the response was measured as sCaw, 1 min after each
Table 1. Details of subjects and treatment in the three studies Study 1: oral
Study 2 : inhaled
oral
amiloride
amiloride
digoxin
Type of subjects
13 asthmatic
24 asthmatic
Sex
Mixed 2 1-68
6 normal, 24 asthmatic Male 18-45 All 8 2
All 14 1
Age range (years) No. taking
Inhaled P-agonists Inhaled steroids Inhaled cromoglvcate
All
4 4
Study 3:
Male
18-45
Sodium transport and bronchial reactivity inhalation, and the test was terminated when s c a w had fallen by 35% from post-saline values. The provocative dose of histamine causing a 20% fall in FEV, (PD2,,FEV,) or a 35% fall in sCaw (PD,,sGaw) was calculated by interpolation on log dose-response plots. Protocols All three studies were double-blind and placebocontrolled. Subjects were instructed to continue their usual treatment throughout the study periods, but to avoid using /3-adrenergic agonists for 6 h before each laboratory visit. All visits for each subject were at the same time of day. Study 1: effect of oral amiloride on bronchial reactivity to histamine. Thirteen asthmatic subjects, aged 2 1-68 years, were studied on 2 days in the same week, ingesting 20 mg of oral amiloride (Morson Pharmaceuticals, Hoddesden, Herts, U.K.) or placebo in randomized crossover fashion. Measurement of FEV, and histamine reactivity (PD,,,FEV, ) was made 2 h after drug ingestion. Study 2: effect of inhaled amiloride on bronchial reactivity to histamine. We studied six normal and 24 asthmatic men, aged 18-45 years. Subjects attended on 2 days within the same week. After baseline measurement of sGaw, subjects received either inhaled amiloride or control diluent in randomized crossover fashion. Drugs were administered via an Inspiron Mini-neb nebulizer (Bard, Sunderland, U.K.) driven by air at 8 I/min. Ten millimetres of 10- mol/l amiloride hydrochloride (Sigma, Poole, Dorset, U.K.) or 10 ml of distilled water (control solution) was placed in the nebulizer chamber and nebulized to dryness. Repeat s Caw measurements were made immediately after inhalation and were followed by a histamine challenge test to measure PD,,sGaw. Study 3: effect of oral digoxin on FEV,, PDz0FEV, for histamine and home peak flow rate. We studied 24 asthmatic men, aged 18-45 years. Subjects attended the laboratory on day 1 for initial assessment and measurement of FEV, and histamine reactivity. They were given a mini-Wright peak flow meter and asked to record twice daily peak flow rate (best of three) for the next 2 weeks. On day 8 subjects returned for a repeat measurement of FEV, and histamine reactivity (PD,,FEV,). They were then randomized to receive either oral digoxin (Wellcome, Crewe, U.K.) 0.5 mg three times daily for 1 day followed by 0.25 mg twice daily for 6 days or matched placebo for 7 days. FEV, ,histamine reactivity and serum digoxin level were measured on day 9 and day 15 (i.e. after 1 and 7 days of digoxin or placebo treatment).
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after the drug between amiloride and placebo in study 2 were compared by using Student's paired t-test. In study 3, changes in FEV, and PD2,,FEVI after digoxin and placebo were compared by analysis of variance. Mean peak flow for weeks 1 and 2 were calculated for each subject and the change in peak flow (week 2 minus week 1) after digoxin and placebo was compared by analysis of variance. P< 0.05 was regarded as significant. All three studies had 95% power to detect differences of one DD of histamine. RESULTS Study 1: effect of oral amiloride on FEV, and bronchial reactivity to histamine The mean (SD) FEV, was 2.8 (0.9) litres and 2.8 (0.7) litres after amiloride and placebo, respectively. The geometric mean (95% confidence intervals) PD2,,FEVI was 0.43 (0.18-1.05) pmol and 0.54 (0.22-1.37) pmol after amiloride and placebo, respectively (Fig. 1). The mean (95% confidence intervals) difference in PD,,,FEV, between amiloride and placebo was 0.34 DD (-0.2 to + 0.9 DD) of histamine ( P = 0.2). Study 2: effect of inhaled amiloride on bronchial reactivity to histamine There was no difference between the placebo and amiloride study days for the mean baseline sCaw, the mean sCaw immediately after inhaled treatment or the change in sCaw after treatment in either the six normal or the 24 asthmatic subjects (Fig. 2). The geometric mean (95% confidence intervals) PD,,sCaw values after amiloride and placebo were 3.0 (1.6-5.7) pmol and 4.3 (2.3-7.9) pmol in the normal subjects, and 0.33 (0.17-0.64) pmol and 0.29 (0.17-0.50) pmol, respectively, in the asthmatic subjects (Fig. 3). The mean (95% confidence intervals) difference in PD,,sGaw between amiloride and placebo was 0.5 ( - 0.53 to + 1.52) DD in the normal subjects ( P = 0.2) and - 0.2 ( - 0.94 to + 0.56) DD in the asthmatic subjects ( P = 0.6).
'"1
Analyses Log-transformed provocative dose values were used in all analyses. For studies 1 and 2, PD2,,FEV, and PD,,sCaw values after drug and placebo were compared by using Student's paired t-test. We expressed the change in PD,,FEV, and PD,,sCaw in terms of DD of histamine. Differences in baseline sGaw and the change in sGaw
0.1
'
I
I
Placebo
Amiloride
Fig. 1. Geometric m e a n k s ~PD2,,FEV, ~ 2 h after oral amiloride or placebo in 13 subjects with asthma.
A. J. Knox et al.
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1.8 1.6
I.% 1.2 "
I
1
I
I
Before
After
Before
After
Fig. 2. Effect of inhaled amiloride ( 0 )or placebo ( 0 )on sCaw in six normal subjects ( u ) and 24 asthmatic subjects ( b ) Values . are means fSEM. Study 3: effect of oral digoxin on FEV,, PD,,FEV, for histamine and home peak flow rate
There was no significant difference between digoxin and placebo in the change in FEV, values after drug (day 8 versus days 9 and 1 S ) , in the change in home peak flow rate after the drug or in the change in PD,,,FEV, after the drug (Figs. 4a and 4b). The geometric mean PDI,,FEV, values on days I , 8, 9 and 15 were 0.7, 0.57, 0.51 and 0.63 pmol o n digoxin, and 0.81, 0.74, 0.89 and 0.86 pmol on placebo (Fig. 4c). The mean (95% confidence intervals) difference in PDI,,FEV, between digoxin and placebo was 0.56 ( - 0.37 to 1S ) DD of histamine after 1 days' treatmcnt ( P = 0 . 2 ) and 0.3 ( - 1.23 to 1.8) DD after 7 days' treatment (1'=0.6). The mean (range) serum digoxin level was 1.1 (0.6-2.5) pg/l after 1 day's treatment and 1.2 (0.6-2.7) pg/l after 7 day's treatment. DISCUSSION
The aim of the present study was to determine whether t w o inhibitors of sodium transport that have been shown to alter airway smooth muscle contractility irz vitro would alter bronchial reactivity in patients with asthma. We studied amiloride which is known to block several sodium transporters including sodium-entry channels found in epithelial tissue, and the Na'/H+ exchanger which is thought to be present in all cells [ 181. Amiloride has also been shown to inhibit Na+/Ca?+ exchange, at lcast in cardiac tissue 119, 201. In studies in bovine trachea irz vitro, we have shown that amiloride inhibits the contractile response to both histamine and carbachol in doses of 10 pmol/l or greater [ 7 ] .Souhrada et al. 1161 have previously shown that in the guinea-pig amiloride attenuates the airway response to antigen in vivo. In study 1, we used the highest recommended dose of oral amiloride but were unable to show any alteration in histamine responsiveness. The diuretic action of amiloride is due to inhibition of epithelial sodium-entry channels and these are blocked by approximately 1 pmol/l concentrations of amiloride [ 181. The plasma concentrations obtained during oral therapy with 5-20 mg of amiloride are slightly greater
.,. .
1
I
Placebo
Amiloride
Fig. 3. Geometric mean fSEM PD,,sGaw after inhaled amiloride or placebo in six normal subjects ( 0 )and 24 asthmatic subjects ( 0 ) .
than 1 pmol/l 1181 and should therefore have blocked sodium-entry channels in airway epithelium in our study. Local drug concentrations in airway smooth muscle may have been lower than those required to inhibit smooth muscle contraction in vitro, however. In study 2, we therefore tried to obtain the maximum possible local concentrations of drug by giving amiloride by inhalation to the limit of its solubility in water ( l o - ' mol/l). The resulting hypo-osmolarity may account for the small, although nonsignificant, reduction in sGaw on both placebo and active treatment days in asthmatic subjects (Fig. 2). The reason we did not see more bronchoconstriction may be due to the fact that we used jet rather than ultrasonic nebulizers 1211. We studied only young atopic men, as the relationship between dietary salt intake and histamine reactivity is stronger in this group of subjects 111, and used PD,,sGaw as a measure of reactivity. since it is more sensitive than PD2,,FEV,and would therefore be more likely to detect small changes 122).Despite this, we again saw no effect of amiloride on histamine responsiveness or sGaw. We have considered whether we achieved adequate tissue concentrations of amiloride. Waltner et ul. [23]have looked at the deposition of inhaled amiloride in normal subjects and patients with cystic fibrosis and showed that a concentration of 10-4 mol/l in airway surface liquid could be achieved by nebulizing 5 x l o - , mol/l amiloride. As we used a concentration of lo-' mol/l, the concentration we achieved in airway surface liquid should have been mol/l. How much of this would approximately 2 x then be absorbed across the bronchial mucosa is uncertain, but it is possible that we were still unable to obtain levels in bronchial smooth muscle of the magnitude required to produce an effect in vitro. Work in sheep suggests that amiloride is taken up into the bloodstream very rapidly after being inhaled, which would further reduce tissue concentrations 1241. It is also possible that an effect on airway smooth muscle was masked by an opposing effect on airway epithelium or inflammatory cells. This would seem unlikely as inhibition of Na' /H ' exchange should reduce inflammatory cell activity [ 12-14] and increase the release of epithelial relaxant
Sodium transport and bronchial reactivity
6001 550
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I
1
2
1
Time (weeks)
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-
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T
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responsiveness [7].This contrasts with a study by Agrawal & Hyatt [ 151 in guinea pigs in vivo, where ouabain caused bronchoconstriction and increased histamine responsiveness. We have previously shown no change in histamine responsiveness in asthmatic subjects after inhaled ouabain [25], but ouabain may not penetrate bronchial mucosa well [26]. We therefore gave oral digoxin in the present study, as it is well absorbed orally and serum concentrations can be measured easily [27]. The serum concentrations we achieved have been shown to produce widespread reduction in tissue Na+/K+-ATPase activity in vivo [28] and it seems likely, therefore, that this occurred in airway smooth muscle in our study. Despite this we did not see any bronchoconstriction or alteration in bronchial reactivity. The reasons for the disparity between these findings and our findings in vitro are not obvious. Digoxin has effects other than Na+/K+-ATPase inhibition, including parasympathomimetic activity, but this would be expected to increase bronchoconstriction. It can also potentiate the effects of circulating catecholamines [27] and this could mask a bronchoconstrictor effect. It is also possible that the Na+/K+-ATPaseinhibition produced by digoxin in vivo was less than that produced by ouabain in vitro. Our studies show that although alterations in cell membrane sodium transport can alter the contractile state of bovine and human airway smooth muscle in vitro, the sodium-transport inhibitors currently available, in the doses we are able to give, d o not alter bronchomotor tone or bronchial reactivity in vivo. Amiloride analogues with greater activity against Na+/H+ exchange and Na+/Ca? exchange than the parent compound have recently been synthesized and might be more effective [29,30]. +
Time (days)
ACKNOWLEDGMENTS We thank the Asthma Research Council for financial support, Dr C. Marenah for measuring serum digoxin levels, and Mr A. Wisniewski and Mrs S. Cooper for technical assistance. REFERENCES
.
0 0
2
4
6 8 1012 Time (days)
14
Fig. 4. Mean k SEM home peak flow rate (PEF ) for weeks 1 and 2 (a), m e a n k ~FEV, ~ ~ (6) and geometric mean k SEM PD,,,FEV, (c) in 24 asthmatic patients before (days 1-7) and after (days 8-15) receiving digoxin ( 0 )or placebo (0).
factor [lo]. Both of these effects should potentiate any relaxant effect of amiloride. In study 3 we studied the effect of digoxin, a Na+/K'ATPase inhibitor, on histamine reactivity. In our studies in vitro, the Na' /K'-ATPase inhibitor ouabain caused contraction of bovine trachea but did not alter histamine
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