Effect of Loop Diuretics on Cholinergic Neurotransmission in Human Airways in Vitro Thorax: First Published As 10.1136/Thx.49.7.657 on 1 July 1994

Thorax 1994;4*.657-663 657

Effect of loop diuretics on cholinergic neurotransmission in human airways In vitro Thorax: first published as 10.1136/thx.49.7.657 on 1 July 1994. Downloaded from

Geert M VerIeden, Jan L Pype, Georges Deneffe, Maurits G Demedts

Abstract induced cholinergc contraction In tiSSUes Background - Frusemide can inhibit with. or without epithelium. various indirectly acting bronchocon- Conclusions - In human airways in vitro strictor stimuli in asthmatic patients. both frusemide and bumetanide pro- Both frusemide and bumetanide also duced a concentration-dependent inhibi- modulate airway neurotransmission in tion of the EFS-induced cholinergic con- some species but there are no data on the tracttion. This inhibition is mediated effect of loop diuretics on neurotransmis- through a prejunctional mechanism. Epi- sion in man. An in vitro study was per- thelium removal was necessary to achieve formed in human airways to investigate this effect. The m anism of action of the possible neuromodulatory action of frusemide and bumeide on airway two loop diuretics, frusemide and bumet- nerves remain unclear: inhlibtion of the anide, and to elucidate whether a cyclo- Na-K-Cl cotransporter is a possibility oxygenase inhibitor such as indometha- and, for frusemide, release of endogenous cmi could modulate the effect of cyclooxygenase products may be involved. frusenmide. The effect of acetazolamide, a Carbonic anhydrase inhibition, on the carbonic anhydrase inhibitor, was also other hand, is unliely to be a factor. investigated. Methods - Electrical field stimulation (Thorax I994,;49-657-663) (EFS; 40 V, 05 ims, 0-5-32 Hz for 15 seconds) in human airways with or without epithelium was used to induce a lIt has recently been shown that frusemide, a cholinergic contraction (n =5 in all ex- loop diuretic, can inhibit various indirectly periments). Indomethacin was present acting bronchoconstrictor challenges in asth- throughout. After obtaining a control matic patients, including exercise,' inhaled http://thorax.bmj.com/ frequency-response curve, different allergen,2 sodium metabisulphite,3 distilled concentrations of diuretic were added to water,4 adenosine,5 and. hype6rtonicsalie6 the organ bath and another frequency- However, frusemide had no inhibitory effect response curve was constructed. To on methacholine or prostaglandin F. induced determine whether the effect of the bronchoconstriction.9 Although this drug diuretic was prejunctional or post- seems to prevent effects of indirect challenges junctional a cumulative concentration- in asthmatics, it is curious that burmetanide, a

response curve to exogenous acetylcho- more potent loop diuretic, causes little or no on September 28, 2021 by guest. Protected copyright. line (Ach, 0-3 pmol/l to 10 mmoI/) was protection against indirect bronchoconstricor constructed in the presence of a diuretic chalenges. Speculation continues, therefore, (frusemide 1 or bumetanide mechanisms Laboratory of mmol/l about the potential of action, Pneumology, 0-1 mmol/l) or its vehicle. In some experi- which may not be related to the diuretic effect Pulmonary ments indomethacim was omitted from of these drugs."r Pharmacology the organ bath to investigate the possible In vitro results point to different sites of Unit, involvement of cyclooxygenase products. Onderwijs en action and these have been reviewed Navorsing Results - Both frusemide (10ltmol/1 to recently."S'2 The in vitro data have come from G M Verleden 1 mmol/l) and bumetanide (1 pimol/I to tissues of different species including guinea J L Pype M G Demedts 01 mmol/l) produced a concentration- pig,'3 equine,'4 and bovine."5 Only a few ex- dependent inhibition of the EFS-induced periments have been performed on human Department of cholinergic contraction in human airways tissue, and no data are available on the possible Thoracic Surgery, in vitro UZ Gasthuisberg but only in epithelium denuded effects of loop diuretics on neurotransmission G Deneffe tissues. Frusemide (1 mmol/l) produced a in human airways in vitro. maximum inhibition of 46-3% (SE 9 9%) We performed this study to assess the effects 3000 Leuven at 0-5 Hz and bumetanide (0-1 mmol/l) 39-6 of frusemide and bumetanide on the choliner- Belgium (6-2)% at 0-5 Hz. Without indomethacin in gic neurotransmission in human airways in Reprint requests to: the organ bath the frusemide-induced vitro. We also investigated the effect of aceta- Dr G M Verleden, UZ Gasthuisberg, inhibition was enhanced at 4, 8, and 16 Hz, zolamide, a carbonic anhydrase inhibitor, on Department of but bumetanide-induced inhibition was cholinergic neurotranssmission as frusemide, Pneumology, 439 Herestraat, B-3000 not enhanced at any frequency when indo- but not bumetanide, possesses an inhibitory Leuven, Belgium. methacin was omitted. Frusemide effect on carbonic anhydrase.' Electrical field Received 29 June 1993 (1 mmol/l) and bumetanide (0-1 mmolfl) stimulation (EFS) was used to release acetyl- Returned to authors 25 August 1993 had no effect on the cumulative concen- choine (Ach) from postiunctional nerves in Revised version received tration-response curve to exogenous Ach human airways and the contractile response to 3 February 1994 Accepted for publication (0-3 imol/l to 10 mmol/1). Acetazolamide exogenous Ach was measured to evaluate a 5 April 1994 (10 mol/l) had no effect on the EFS- possible neuromodulatory effect. 658 Verleden, Pype, Deneffe, Demedts Methods which gives a maximal inhibitory effect. Only one concentration of diuretic was added per tissue. After 10 minutes incubation a third TISSUE PREPARATION frequency-response curve was performed. Human airways were obtained from 35 The responses to EFS in vehicle-treated tis- Thorax: first published as 10.1136/thx.49.7.657 on 1 July 1994. Downloaded from patients (33 men, two women) of mean age sues were stable throughout the period of the 63-6 (range 43-76) years undergoing pulmon- experiment. Six tissues were simultaneously ary resection, mostly for bronchial carcinoma. studied with at least one time control tissue per Immediately after surgery the airways were experiment. The responses to EFS were com- macroscopically dissected in the operating pletely blocked by tetrodotoxin (1 pmol/l), theatre, placed in cold (4°C) Krebs-Henseleit confirming their neuronal origin. (KH) solution (NaCl 118, KC1 59, MgSO4 1-2, CaCl2 2-5, NaH2PO4 1 2, NaHCO3 25-5, Acetylcholine concentration-response curve and glucose 5-5 mmol/l) and transported to the The effect of frusemide (1 mmol/l) or bumeta- laboratory. The airways were carefully nide (100 jimol/l) on a cumulative concentra- stripped from surrounding lung tissue and cut tion-response curve to exogenous Ach into strips (main bronchi) or ring segments (0 3 j.mol/l to 10 mmol/1) was studied in hu- (segmental and subsegmental bronchi) which man bronchi after a 10 minute incubation were mounted between two platinum wire period. The results were expressed as a per- electrodes in 10 ml organ baths containing KH centage of the maximum contractile response solution at 37°C and continuously bubbled to Ach (1Ommol/l) which was determined at with 95% oxygen and 5% carbon dioxide. the beginning of the experiment. Indomethacin (10 jmol/1) was present in the organ bath throughout the experiment to block DRUGS the formation of prostaglandins which may The drugs used in these experiments were interfere with neutrotransmission.'7 In a dif- obtained from the Sigma Chemical Co (Eupen, ferent series of experiments indomethacin was Belgium). Stock solutions of frusemide were omitted from the organ bath to investigate the made up in 100 pmol/l sodium hydroxide and possible involvement of cyclooxygenase pro- further diluted with distilled water. Stock ducts. solutions of bumetanide were made up in In most experiments the epithelium was 100% ethanol and further diluted with ethanol removed by gently rubbing the luminal surface to the appropriate concentration. Acetazola- of the bronchi for two minutes with a cotton mide was dissolved in 100 pmol/l sodium hy- wool applicator. Complete removal of the epi- droxide and further diluted with distilled thelium was checked by light microscopy by an

water. All control responses were obtained in http://thorax.bmj.com/ experienced pathologist in a blind fashion. the presence of an equivalent amount of sol- Bronchial strips or rings were connected via vent. Indomethacin was made up in alkaline silk threads to Grass FT-03 force displacement phosphate buffer (KH2PO4 20 mmol/l, transducers (Grass Instruments, Quincy, Na2HPO4 120 mmol/l; pH 7-8). Fresh drug Massachusetts, USA) to measure isometric solutions were made up daily. Drug additions changes in tension. The tissues were allowed did not exceed 1% of the bath volume. All to equilibrate for one hour whilst being concentrations refer to the final organ bath washed with fresh KH solution every 20 concentrations. minutes under a resting tension of 2g, which on September 28, 2021 by guest. Protected copyright. was found to be optimal for measuring changes in tension.'8 EFS was produced by a Harvard ANALYSIS OF RESULTS student stimulator (Harvard, Kent, UK). All contractile responses were measured as the Biphasic square wave pulses of a supramax- difference between peak tension and the rest- imal voltage of 40 V at source and a pulse ing tension that developed. The effect of the duration of 0 5 ms were applied for 15 seconds diuretic was expressed as percentage inhibi- every four minutes at frequencies ranging tion, comparing the contractile responses at from 0 5 to 32 Hz. Isometric contractile re- each frequency after pretreatment with the sponses were visualised on a computer screen diuretic with the contraction at the same fre- after digitalisation of the signal (Codas, Dataq quency in the control response. Since each Instruments, Ohio, USA) and recorded on a tissue acted as its own control analysis of data personal computer. was possible using a Student's t test for paired data. Contractile responses to exogenous Ach were compared between diuretic-treated and PROTOCOL vehicle-treated tissues by the Student's t test Electrical field stimulation for unpaired data. Results are expressed as In human airways EFS causes a rapid contrac- mean (SE), p values of < 0 05 being con- tion that can be abolished by pretreatment of sidered significant. the tissue with atropine, confirming that the contractile response is due to Ach release. After a one hour period of equilibration a Results frequency-response curve was constructed EFS in human airways (40 V, 0 5 ms, 0 5- and discarded. After washing the tissues a 32 Hz for 15 seconds) resulted in a rapid cho- control frequency-response curve was per- linergic contraction which increased with formed after which a diuretic or its vehicle (in increasing frequencies of stimulation. A typi- a separate tissue) was added to the organ bath. cal trace, which also demonstrated the inhibit- The diuretics were incubated for 10 minutes ory effect of frusemide, is shown in fig 1. The Modulation of cholinergic neurotransmission by loop diuretics 659 inhibitory effect of frusemide (100 imol/l) in absolute measurements is shown in the table.

EFFECT OF FRUSEMIDE ON CHOLINERGIC Thorax: first published as 10.1136/thx.49.7.657 on 1 July 1994. Downloaded from RESPONSES In tissues with epithelium frusemide (1 mmol/ 1) produced no inhibition of the cholinergic contractions to EFS, with or without indomethacin (10 pimol/l). When the epithelium was removed and indomethacin (10 iimol/l) was added to the organ bath, frusemide (100 gmol/l and 1 mmol/l) inhibited the cho- 0.5 1 2 4 8 16 32 Hz linergic contractile responses at all frequencies, which was, however, not significant at 32 Hz. Frusemide (10 jimol/l) had no more significant inhibitory effect on the cholinergic responses. Frusemide (1 mmol/l) produced a maximum inhibition of 46-3 (9 9)% (n=5, p < 0 05) at 0 5 Hz and 45 (4-6)% at 1 Hz Frusemide (1 mmol/l) stimulation (n =5, p <0-001). There was no significant effect of the vehicle on the cholinergic responses (fig 2). F Without indomethacin in the organ bath frusemide (10 pmol/l) produced no inhibition * * * U 4 8 16 32 Hz of the cholinergic responses. The inhibition 0.5 produced by 100 ,imol/l frusemide, however, Figure I Effect of electrical field stimulation (EFS) (40 V, 0 5 ms, 0 5-32 Hz for was significantly enhanced at 4, 8, and 16 Hz 15 seconds) in human airways (U). Frusemide (1 mmol/l) clearly inhibited the cholinergic contraction at all frequencies. stimulation; at 8 Hz, for example, frusemide (100 pmol/l) produced an inhibition of 12-5 (2-9)% with indomethacin (10 JLmol/l) and 27-4 (2-3)% without (n = 5, p < 001) (fig 3). Mean (SE) inhibitory effect offrusemide (IOQumolll) The responses to exogenous Ach (0 3 jimol/l in absolute measurements (mg) to 10 mmol/l), with or without indomethacin, Frequency were not significantly altered by frusemide

(Hz) Control Frusemide % inhibition (1 mmol/l) (fig 4). http://thorax.bmj.com/ 0-5 302(78) 211(58) 30 2(5) 1 327(69) 223(51) 31.9(4) 2 463(71) 299(63) 35-5(4.6) 4 618(91) 516(73) 16-6(2) EFFECT OF BUMETANIDE ON CHOLINERGIC 8 809(72) 717(89) 12 5(2 9) RESPONSES 16 1064(99) 986(108) 7-4(1 1) 32 1358(109) 1238(73) 8-8(2-1) In tissues with epithelium bumetanide (100 pmol/l) had no inhibitory effect on the EFS-induced cholinergic contractions. When the was removed bumetanide (10

epithelium on September 28, 2021 by guest. Protected copyright. 60 and 100 pmol/l) inhibited the cholinergic re- r * sponses at all frequencies, but not significantly at 16 Hz and 32 Hz for 100 gimol/l bumetanide. 50 F- Bumetanide (100 pmol/l) produced a maximum inhibition of 39-6 (6-2)% at 0 5 Hz 40 k (n= 5, p < 0 01) and 38-4 (6-6)% at 1 Hz stimulation (n= 5, p < 0 05). At 1 gmol/l bumetanide c 30 H had no significant inhibitory effect on the cho- 0 linergic responses, and no significant effect was '- 20 H seen with the vehicle for bumetanide (fig 5). When indomethacin (10 imol/l) was omit- -0 ted the inhibition produced by bumetanide 10 | (10 imol/l) was similar to that produced by the same concentration of bumetanide in the pres- 0 ence of indomethacin (10 Jmol/l) (fig 6). The responses to exogenous Ach (0 3 gimol/l -10 _ to 10 mmol/l) were not affected by bumetanide (100 jimol/l) (fig 7). -20 _

0.5 1 2 4 8 16 32 EFFECT OF ACETAZOLAMIDE ON CHOLINERGIC Frequency (Hz) RESPONSES In tissues with or without epithelium actezola- Figure 2 Inhibition of EFS-induced cholinergic contraction by different concentrations mide (100 ,mol/l) produced no significant offrusemide in human epithelium-denuded airways in the presence of indomethacin inhibitory effect (2-2(1-5)%, n= 5) on the cho- (10 umol/l):@, 10,wmol/lfrusemide; A, 100 pmol/l frusemide; U, I mmol/lfrusemide. The frusemide vehicle (0) had no effect on the cholinergic contractions. Means (SE) linergic responses to EFS in human bronchi values offive tissues of different patients are shown. ***p < 0-001, **p < 0.01, *p < 0.05. (data not shown). 6w Verleden, Pype, Deneffe, Demedts 60 was enhanced, although not significantly at all frequencies. This suggests that cyclooxygenase products may be involved in the mechan- 50 ism of action of frusemide. Acetazolamide, a carbonic anhydrase inhibitor, produced no Thorax: first published as 10.1136/thx.49.7.657 on 1 July 1994. Downloaded from inhibition of the cholinergic contraction. 40 - The mechanism of action of frusemide is not c 0 fully understood. In our experiments both .0 frusemide and bumetanide inhibited the cho- 30 - linergic contraction to EFS, so inhibition of \* the Na-K-Cl cotransportor, which is one of the mechanisms of action of loop diuretics in 20 - the kidney,19 cannot be excluded. Moreover, bumetanide was found to be about 10 times more potent than frusemide, as has been 10 _- shown in previous in vitro studies.1320 In in vivo challenges bumetanide has little or no effect.' The same is true for piretanide and 0 toresamide which are more potent diuretics but have less protective effect than frusemide 0.5 1 2 4 8 16 32 in bronchoconstrictor challenges in asthmatic Frequency (Hz) patients.2' Some investigators therefore ques- Figure 3 1, hibition of EFS-induced cholinergic contraction in epitheium-denuded tcon whether the inhibition of theNa-K-tCl tissues byfmsemide (IOO mooX/1) in the presence (A) and absence (I]) of contransporterisapossibleexplanationforthe indomethacit_ (10 _ltl). Indomethacin attenuated the inhibitory effect of fruseinde. anti-asthmaticeffcects of frusemide.10 How- Mean (SE) values offive tissues are shown. **p < 0-01, *p < 0-05. ever, our results in human airways in vitro indicate that this effect certainly can not be dismissed, and the discrepancy between in 100 r vivo and in vitro results remains difficult to interpret. It has been postulated that local 90 V pharmacokinetic effects in the airways in vivo 1- might explain this discrepancy and, in fact, 80 frusemide, but not bumetanide, has been C- 70 - shown to produce an acute elevation of plasma 00 renin, and an increased peripheral blood Om E 60 V flow,22 which could account for an increased http://thorax.bmj.com/ c- 0 0o-- washout of exogenously administered bron- 50 choconstrictor agents. Moreover, bumetanide, 0 in contrast to frusemide, is highly lipid solu- ._ cr m 40 ble, suggesting that bumetanide is not main- c . >.0 30 - tained in a high enough concentration at its site

O 0X of action in the airways in Vivo.21 ,,o Co 20 _ Another explanation for the different effects of frusemide and bumetanide in vivo could be on September 28, 2021 by guest. Protected copyright. 10 the fact that frusemide also possesses an inhibitory effect on carbonic anhydrase activity o alr | | which bumetanide does not share, and that 6 5 4 3 2 frusemide exerts its effect through inhibition of this enzyme.' Our experiments, however, failed to demonstrate an inhibitory effect of Figure 4 C,umulative concentratin-response curve to exogenous acetylcholine acetazolamide on the EFS-induced cholinergic (0-3 umol/l Jto 10 mmol/l) in the presence (0) and absence (0) offrusemide contraction, which makes this possibility (1 mmol/l). Mean (SE) values offive different epithelium-denuded tissues are shown. unlikely. Moreover, carbonic anhydrase inhibition is unimportant in vitro as bumetanide, which has no such activity, was more effective than frusemide in our experiments. Loop diuretics have an effect on airway Discussion nerves seen in guinea pig bronchi"24 and tra- These results show that the loop diuretics chea2w in vitro. Frusemide also has an inhibit- frusemide and bumetanide inhibit the cho- ory effect on various inflammatory cells, and linergic contractions in response to EFS in the latter mechanism is probably important in human airways in vitro in a concentration some challenges in asthmatic patients. Fru- dependent manner. Removal of the epithelium semide inhibits the release of histamine and was necessary to achieve this effect. Neither leukotrienes in passively sensitised human diuretic, however, affected the cumulative lung in vitro25 and in actively sensitised guinea concentration-response relationship to exo- pig lung.26 It also inhibits mediator release genous Ach, suggesting that they may act from eosinophils in vitro' by inhibition of through a prejunctional mechanism. When chloride transport. From these findings it has indomethacin was omitted from the organ bath been suggested that frusemide exerts its effects inhibition of the cholinergic contraction through inhibition of a specific chloride chan- induced by frusemide, but not by bumetanide, nel."' This seems an attractive hypothesis as Modulation of cholinergic neurotransmission by loop diuretics 661 60 r been shown to modulate airway neurotransmission.'7 30 Recent data provide evidence 50K that inhibitory prostaglandins are involved in the prevention of exercise-induced asthma by 40 V frusemide. Indomethacin (50 mg three times Thorax: first published as 10.1136/thx.49.7.657 on 1 July 1994. Downloaded from daily) attenuated the protective effect of fruse- c 30 F- mide on exercise-induced asthma,3' and flurbi- 0 profen, another cyclooxygenase inhibitor, did .0 20 F- not modulate the protective effect of frusemide CE on metabisulphite-induced bronchoconstriction. This is either evidence against the release 10- of inhibitory prostaglandins by frusemide,32 or +--i* * for the release of prostaglandins by frusemide 0 under certain conditions such as exercise- induced asthma or other mast cell degranulat- -10 F- ing stimuli. Others have also failed to show increased production of prostaglandin E2 in -20 '- human nasal mucosa in vivo after frusemide instillation.33 In our experiments the fruse- I mide-induced inhibition of the cholinergic 0 5 1 2 4 8 16 32 contraction was enhanced when indomethacin Frequency (Hz) was omitted from the organ bath, although this Figure 5 Inhibitory effect of different concentrations of bumetanide on the enhancement was only seen with a high dose EFS-inducced cholinergic contractions in human epithelium-denuded airways in the (100 pmol/l). The bumetanide-induced inhibi- presence ofIindomethacin (10 mol/l): *, I ,umol/l bumetanide; A, 10Oumol/l tion was, however, not enhanced when indo- bumetanidee; U, 100 pmol/l bumetanide. The bumetanide vehicle (A) had no effect on methacin was omitted from the organ bath, the choline,rgic contractions. Mean (SE) values offive different tissues are shown. perhaps because it has no inhibitory effect on **p< 01, *p<0.0S. 1 5-hydroxyprostaglandin dehydrogenase, an enzyme involved in the inactivation of prostaglandins.34 Indomethacin in a concentration of 10 pmol/l augments the responsiveness of 50 r unrubbed canine bronchial rings to acetylcholine, which may be due to inhibition of the C generation of prostaglandins.35 These results 0 suggest that the release of prostaglandins may C., 40 F- http://thorax.bmj.com/ co-0 be involved in the inhibition induced by frusemide, but not by bumetanide, although this c)0 effect not be Other in vitro C.) may important. IL) 30 K experiments also suggest an influence of inhi- Co C- bitory prostaglandins.36 In our experiments loop diuretics only produced inhibition of the cholinergic contrac- 0 20 tions in tissues without epithelium and it C seems that these on September 28, 2021 by guest. Protected copyright. .00 unlikely inhibitory prosta- 0C noids are released by the epithelium. The site -0 of prostaglandin release therefore remains 10I speculative. -O Frusemide failed to produce an inhibitory effect on cholinergic contractions in equine 0 tracheal tisue without epithelium, suggesting i e I that it produces its effects in this species 0.5 1 2 4 8 16 32 through release of an epithelium-dependent Frequency (Hz) relaxant factor.'4 This is in agreement with the effect of frusemide on vascular smooth muscle Figure 6 Inhibition of the EFS-induced cholinergic contraction in epithelium- denuded tisssues by bumetanide (10 jumol/l) in the presence (0) and absence (a) of where the vasoconstrictor response to pen- indomethae,in (10 pmol/l). Indomethacin withdrawal did not result in an increased arterial electrical stimulation in rat tail artery inhibitory ieffect of bumetanide. Mean (SE) offive tissues are shomn. with intact endothelium was inhibited, but this effect was absent in endothelium-denuded preparations.37 This contrasts with the findings of the previous studies - for example, nedocromil sodium possesses an inhibitory Elwood et al'3 showed that frusemide modu- effect on chloride transport,2829 but whether lated the cholinergic and non-cholinergic con- chloride channel blockers can inhibit cholin- tractions in guinea pig airways by a prejunc- ergic contraction in human airways in vitro tional mechanism which was independent of remains to be investigated. the epithelium. In the present study, however, Frusemide enhances the synthesis of prosta- epithelium removal was necessary to achieve glandin E2 in the kidney and it has therefore an inhibition of the cholinergic contraction by been postulated that prostaglandin release may frusemide and bumetanide. be involved in the protective effects of fruse- Mechanical removal of the epithelium en- mide. Indeed, prostaglandins El and E2 have hances the responsiveness and sensitivity to 662 Verleden, Pype, Deneffe, Demedts

1 Bianco S, Vaghi A, Robuschi M, Pasargiklian M. Preven- tion of exercise-induced bronchoconstriction by inhaled furosemide. Lancet 1988;ii:252-5. 2 Bianco S, Pieroni MG, Refini RM, Rottoli L, Sestoli P. Protective effect of inhaled furosemide on allergen-

induced early and late asthmatic reaction. N EnglJfMed Thorax: first published as 10.1136/thx.49.7.657 on 1 July 1994. Downloaded from 1989;321:1069-73. C- 3 O'Connor BJ, Chung KF, Chen-Worsdel YM, Fuller RW, 00 Barnes PJ. Effect of inhaled furosemide and bumetanide * E 80 on adenosine 5'-monophosphate and sodium metabisul- E fite-induced bronchoconstriction in asthmatic subjects. Am Rev Respir Dis 1991;143:1329-33. co,-0 4 Robuschi M, Gamboro G, Spagnotto S, Vaghi A, Bianco S. Inhaled furosemide is highly effective in preventing ultrasonically nebulised water bronchoconstriction. Pulm 60 Pharmacol 1989;1:187-91. 5 Polosa R, Lau LC, Holgate ST. Inhibition of adenosine 5'- monophosphate- and methacholine-induced bronchocon- 40 striction in asthma by inhaled furosemide. Eur Respir J 1990;3:665-72. 6 Rodwell LG, Anderson SD, du Toit JI, Seale JP. The 0 effect of inhaled frusemide on airway sensitivity to inhaled 4-5% sodium chloride aerosol in asthmatic subjects. 20 Thorax 1993;48:208-13. 7 Nichol GM, Alton EWFW, Nix A, Geddes DM, Chung KF, Barnes PJ. Effect of inhaled furosemide on metabi- sulfite and methacholine induced bronchoconstriction 0 and nasal potential difference in asthmatic subjects. Am Rev Respir Dis 1990;142:576-80. 8 Grubbe RE, Hopp R, Dave NK, Brennan B, Bewtra A, 6 5 4 3 2 Townley R. Effect of inhaled furosemide on the bronchial response to methacholine and cold-air hyperventilation -log [Acetylcholine (mol/ll)] challenges. J Allergy Clin Immunol 1990;85:881-4. 9 Stone RA, Yeo TC, Barnes PJ, Chung KF. Frusemide Figure 7 Cumulative concentration-response curve to exogenoussacetylcholine inhibits cough but not bronchoconstriction to prosta- (0-3 umol/l to 10 mmol/l) in epithelium-denuded tissues in the presence and absence glandin F2, in patients with asthma (abstract). Am Rev (0) Dis 1991;143:A548. (0) of bumetanide (100 pmol/l). Mean (SE) values of eight tissues are shown. 10 BarnesRespirPJ. Diuretics and asthma. Thorax 1993;48:195-6. 11 Chung KF, Barnes PJ. Loop diuretics and asthma. Pulm Pharmacol 1992;5:1-7. 12 Bianco S, Pieroni MG, Refini RM, Robuschni M, Vaghi A, Sestini P. Inhaled loop diuretics as potetial new anti- several spasmogens in airway preparations asthmatic drugs. Eur Respir J 1993;6:130-4. from several animal specives including man.38 13 Elwood WE, Lotvall JO, Barnes PJ, Chung KF. Loop diuretics inhibit cholinergic and noncholinergic nerves in This increased reactivity may be due to the guinea pig airways. Am Rev Respir Dis 1991;143:1340-4. existence of an inhibitory signal generated by 14 Yu M, Wang Z, Robinson NE, Derksen FJ. The inhibitory effect of furosemide on the contractile response of equine the epithelium."5 The losss of an inhibitory trachealis to cholinergic nerve stimulation. Pulm Pharma- factor cannot explain the inhibitory effect of col 1992;5: 233-8. 15 Knox AJ, Ajao P. Effect of furosemide on airway smooth loop diuretics in epitheliurm-denuded human muscle contractility in vitro. Thorax 1990;45:856-9. 16 Ward AB, Heel RC. Bumetanide: a review of its pharmaco- http://thorax.bmj.com/ airways in vitro. dynamic and pharmacokinetic properties and therapeutic Epithelium removal restults in loss of a bar- use. Drugs 1984;28:426-64. rier function.39 A direct effect of 17 Ito Y. Prejunctional control of excitatory neuroeffector inhibitory transmission by prostaglandins in the airway smooth frusemide on the smooth muscle is unlikely, muscle tissue. Am Rev Respir Dis 1992;143:S6-S10. however, as it has no effecrt on smooth muscle 18 Belvisi MG, Stretton CD, Verleden GM, Ledingham SJL, Yacoub MH, Barnes PJ. Inhibition of cholinergic neuro- contractions induced by various constrictor transmission in human airways by opioids. J ApplPhysiol agents.15 This was also skeen in the present 1992;72:1096-100. 19 O'Grady SM, Palfrey HC, Field M. Na-K-2Cl cotransport study as frusemide did nlot affect the dose- in winter flounder intestine and bovine kidney outer

curve to Ach. medulla; 3[H] bumetanide binding and effects of furo- on September 28, 2021 by guest. Protected copyright. response exogenotus semide analogues. J Membr Biol 1987;98:11-8. Epithelium removal al[so exposes airway 20 Verleden GM, Pype JL, Demedts MG. The effect of nerves, a state similar to tthe airways of asth- furosemide on non-adrenergic relaxation in guinea-pig trachea in vitro. Eur Respir J 1992;5(Suppl 15): 167s. matic subjects, where ch,ronic inflammation 21 Foresti A, Pelucchi A, Mastropasqua B, Cavigioli G, Ear- causes damage to the )ithelium, exposing lesi RM, Marazzini L. Effect of inhaled furosemide and ep By . . toresamide on bronchial responses to ultrasonically sensory nerve endings.-'41 By removing the nebulized distilled water in asthmatic subjects. Am Rev and a nerves fruse- Respir Dis 1992;146:364-8. epithelium exposing irway 22 Passmore AP, Whitehead EM, Johnston GD. Comparison mide could readily reach thie nerves to exert its of the acute renal and peripheral vascular responses to frusemide and bumetanide at flow and high dose. Br J inhibitory effect. As higb1 concentrations of Clin Pharmacol 1989;27:305-12. frusemide are needed to in] iibit the cholinergic 23 Lant A. Diuretics: clinical pharmacology and therapeutic contraction, however, a ,ssible toxic effect of use. Drugs 1985;29:57-87. po 24 Mapp CE, Boniotti A, Papi A, Maggi CA, Di Stefano A, the loop diuretics on the aiirway nerves cannot Saetta M, Ciaccia A, Fabbri LA. Effect of bumetanide on be excluded. toluene diisocyanate induced contractions in guinea-pig airways. Thorax 1993;48:63-7. This study has shown thLat loop diuretics, in 25 Anderson SD, Wei HE, Temple DM. Inhibition by furo- a inhibit the semide of inflammatory mediators from lung fragments. concentration-dependent manner, N Engl J Med 1991;324:131. cholinergic contraction in human airways in 26 Berti F, Rossoni G, Zuccari G, Buschi A, Robuschi M, a m but Villa LM, et al. Protective activity of inhaled furosemide vitro by prejunctional echanism, only against immunological respiratory changes and mediator in epithelium-denuded tissues. Cyclooxygen- release in guinea-pigs. Pulm Pharmacol 1992;5:115-20. ase products enhance thiis inhibitory effect 27 Perkins R, Dent G, Chung KF, Barnes PJ. Effect of anion transport inhibitors and extracellular Cl-concentrations with frusemide, whilst a (carboniccarbonicanhyclraseanhydrase on eosinophil respiratory burst activity. Biochem Pharma- inhibitor has no inhibitory effect at all. Our col 1992;107:481-8. experiments suggest that i:nhibition of Na-K- 28 JacksonnedocromilDM, sodiumPollardonCE,theRobertsisolated SM.rabbitThevaguseffectnerve.of Cl cotransporter may exp lain the Eur J Pharmacol 1992;221:175-8. inhibitory 29 Reinsprecht M, Pecht I, Schindler H, Romanin C. Potent effect of loop diuretics on ccholinergic contrac- block of Cl-channels by antiallergic drugs. Biochem Bio- tion. However, there is no as to phys Res Commun 1992;188:957-63. explanation 30 Aikawa T, Sekizawa K, Itabashi S, Sasaki H, Takashima T. why epithelium removal Iis necessary tto Inhibitory action of prostaglandin E, on non-adrenergic, achieve this effect in human in vitro. non-cholinergic contraction in guinea-pig bronchus. Br J airways Pharmacol 1990;101:13-4. JLP is funded by Allen-Glaxo (Belggium). 31 Pavord ID, Wisniewski A, Tattersfield AE. Inhaled fru- Modulation of cholinergic neurotransmission by loop diuretics 663

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Adventitia

A Philadelphia summer

"The August heat was merciless. From the been found in the bodies of fatal cases and a seventh to the twenty-seventh there were only left wing group (perhaps communist sailors two cool days. Twice there was rain and after- from a Polish ship moored in the Delaware wards the sun shone through a steamy mist. river) were thought to have injected nickel Those who have lived through Philadelphia carbonyl gas into the air conditioning systems summers know these afternoons and would of hotels where the legionnaires were staying. view with compassion the moist faces of the Or was there some noxious element in the delegates". Thus wrote William Johnson, a droppings of those rabbits and doves conjured Connecticut delegate to the Constitutional out of hats at the magicians' convention? Convention which followed the Declaration of These rumours were not confined to the http://thorax.bmj.com/ Independence two centuries ago. tabloids and taverns of Philadelphia. Even the Things were much the same in August 1976. Lancet, as late as June 1977, was still promot- The sweltering city of the founding fathers ing the nickel theory and contemplating the was again host to a great variety of groups possibility that "careless chemists," "mad celebrating the bicentenary of independence. scientists," "saboteurs," or "criminals" were There were conventions of magicians and responsible; or perhaps the nickel carbonyl gas candlemakers, a Eucharistic congress, national was coming from the garbage cans in which

archery and rowing championships - and the Philadelphians were burning their rubbish on September 28, 2021 by guest. Protected copyright. fall meeting of the American Physiological because of a garbage collectors' strike? And so Society to which I was to present a paper. the theories proliferated until Joseph McDate We had one great advantage over the per- noticed a curious red patch on a culture from a spiring delegates of 200 years ago - air condi- guinea pig inoculated with lung tissue from a tioning - or did we? The first hint that some- dead legionnaire. From this he grew Legionella thing was amiss was when they switched off pneumophila, the Gram negative bacillus now the air conditioning at my hotel. I was assured known to be the cause of the outbreak. that this was a routine servicing procedure - Of 182 patients with legionella pneumonia, but why in the middle of an August heat wave 29 died. The victims included 149 legion- and who were those heavies snooping around naires, nine Eucharists, two candlemakers, and the lobby and corridors? And then the story one magician - but no physiologists. The broke with screaming headlines in the Phila- majority of these had - as indeed had I - delphia Daily News: dozens of delegates to the entered the Bellevue-Stratford hotel or convention of the Pennsylvania branch of the strolled along the adjacent sidewalk on Broad American Legion, held in the city two weeks Street. Raised serum titres among the hotel earlier, were being smitten by a mysterious employees suggested long term exposure to the disease from which 19 had already died. Hotel bacillus and a degree of immunity, for only one bookings were cancelled and the city, which of the 400 employees contracted the disease. had invested billions of dollars in the bicenten- This solitary case was the air conditioner man. nial celebrations, was facing bankruptcy. Perhaps, like our famous predecessors two Rumours abounded. The veterans of foreign centuries ago, we too should have sweated out wars accused drug companies of conspiring to that Philadelphia summer and eschewed the release a fatal flu virus in order to promote comforts of modern technology. their vaccine sales. High levels of nickel had COLIN OGILVIE

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