Eur Respir J 1993, 6, 130- 134 SHORT REVIEW

Inhaled loop diuretics as potential new anti-asthmatic drugs

S. Bianco*, M.G. Pieroni*", R.M. Refini**, M. Robuschi+, A. Vaghi++, P. Sestini**

Inhaled loop diuretics as potential new anti-asthmatic drugs. S. Bianco, M.G. Pieroni, R.M. Refini, M. Robuschi, A. Vaghi, P. Sestini .

* Institute of Respiratory and Cardio­vascular Diseases, Ospedale S. Raffaele, Milan, Italy. ** Institute of Respiratory Diseases, University of Siena, Italy. • Institute of Respirato ry Diseases, University of Milan, Italy. ++ Ospedale di Garbagnate, Milan, ftaly.

ABSTRACT: The observation that changes in bronchial osmolari ty can induce bronchoconstriction in asthma inspired the experimental studies which, unex­pectedly, revealed that fr usemide is an effective bronchoprotective agent against a variety of osmotic and non osmotic stimuli. Although the mechanism of this protective effect is not fully understood, s tudies in vivo and iiZ vitro suggest that frusemide may inhibit tbe activation of different cell types induced by bron­choconstrictor stimuli. Other loop diuretics also exert bronchoprotective activ­ity, but frusemide appears to be the mor e effective bronchoprotective agent of this family, r egardless of their diuretic potency and lipid solubility. Despite the relatively large amount of experimental evidence, there is currently lit tle infor­mation on the clinical effectiveness of frusemide in asthma and a long-term con trolled study is currently in progress. T he observations that treatment with a combination of inhaled acetylsalicylate and frusemide results in a markedly increased bronchoprotective effect compared to either drug a lone, opens a new perspective in the possible clinical use of these drugs. Prelimina ry studies sug· gest that the association of these drugs is well tolerated and may result in a remarkable steroid sparing effect in patients with steroid dependent asthma, for whom a convenient alternative to long-term steroid therapy is not currently availa ble.

Correspondence: S. Bianco Institute of Respiratory and Cardiovas­cular Diseases Ospedale S. Raffaele Milan Italy

Keywords: Asthma bronchial hyperreactivity frusemide loop d iuretics

Received: June 25, 1992 Accepted after revision October 4, 1992

Eur Respir J., 1993, 6, 130- 134.

Introduction

The physicochemical characteristics of the fluid lining the airways affect bronchial reactivity in asth­matic patients, as indicated by the bronchoconstriction induced in these patients by a variety of stimuli that affect the osmolarity of the bronchial environment [I]. The liquid and ion composition of the bronchial lining fluid is largely regulated by ion transport path­ways in the epithelial cells of the airways [2, 3]. The original idea behind the series of acute studies on the effect of frusemide on bronchial reactivity was that, since loop diuretics inhibit the basolateral Na+/l(+/Cl· eo-transport in epithel.ial cells [4, 5], this effect could change the bronchial response to osmolar stimuli. Unexpectedly, these experiments demonstrated that inhaled frusemide is a very effective broncho­protective agent and, hence, a potential anti-asthmatic drug.

The aim of this paper is to summarize the results obtained using inhaled frusemide and other loop diu­retics against a variety of bronchoconstrictor stimuli in asthmatic patients, to examine the possible mechanism of action of the bronchial protective effect of these drugs, and to consider the possible future application of these drugs in the therapy of asthma.

Effect of inhaled frusemide on experimentally­induced bronchoconstriction

Osmolar stimulants were the first bronchoconstric­tor agents against which the effect of inhaled fruse­mide was tested. In a series of controlled studies we found that inhalation of 40 mg frusemide inhibited the bronchoconstrictor response to ultrasonically nebulized water [6, 7], exercise [8], and hypertonic solutions of NaCl [9]. Under the same experimental conditions, oral frusemide was ineffective, suggesting that the protec­tive effect was due to direct action of inhaled fruse­mide on the bronchial mucosa. More recently, inhaled frusemide has also been shown to protect asthmatic patients against cold air and hypertonic KCJ-induced bronchoconstriction [ 10, 11], and against blood gas changes induced by ultrasonically nebulized distilled water [12]. The nature of the osmotic changes in­duced in these models varies considerably, depending on the stimulus: hypotonic aerosols, such as distilled water, are presumed to reduce bronchial osmolarity, whereas hypertonic solutions, exercise and cold air hyperventilation are presumed to have the opposite effect. The fact that significant protection has been observed against all of these stimuli suggests that in­haled frusemide does not counteract the osmotic

INHALED LOOP DIURETICS IN ASTHMA 131

changes directly, but probably interferes with mecha­nisms triggered by these changes, such as stimulation of sensory receptors and/or release of mediators from superficial airway mast cells [13].

The release of preformed and newly generated me­diators from superficial bronchial mast cells is very relevant to the bronchial response to allergens [14]. We speculated that if the protective activity of fruse­mide was, at least partially, mediated by a stabiHzing effect on these cells, then frusemide could also inhibit the immediate bronchoconstrictor response to allergens. In controlled studies, inhaled frusemide protected asthmatic subjects from the early and late responses to allergen challenge [15-19], but not from the increase in bronchial reactivity following the late re­action [ 18]. Inhaled frusemide was also found to reverse the early response, when administered after allergen chaHenge [18]. The protective effect observed on the late response [19] was also of interest, because the time of its occurrence, 4-8 h after the challenge, frusemide is beyond the estimated duration of action of inhaled frusemide. According to preliminary stud­ies on the distilled water-induced airway response, furosemide is most active in the first hour after ad­ministration [20]. Tn a single study, no further pro­tection against the late response was afforded by a second dose of frusemide administered two hours af­ter challenge (unpublished results). Frusemide presum­ably exerts its protective activity during the early phase of the reaction, by inhibiting the release of in­flammatory mediators necessary for the development of the late response.

In patients with allergic rhinitis, premedication with frusemide has also been shown to prevent the increase in bronchial responsiveness to methacholine that follows the early asthmatic response [I 7], a finding which we were unable to confirm in a subsequent study in asthmatic patients [2 I]. Actively sensitized guinea-pigs [16J, and dogs [22], were also protected by inhaled frusemide against bronchoconstricrion in­duced by antigen inhalation but not by nonspecific stimuli. An intravenous infusion of frusemide fai led to protect guinea-pigs from the anaphylactic response, suggesting that in this model the drug also acts via a local mechanism [I6].

Frusemide also gives significant protection against challenge with sodium metabisulphite and adenosine [23- 25]. The mechanism of action of these bron­choconstrictor agents is still unknown, but is consid­ered to be, at least partially, mediated by nerve and mast cell activation. In contrast, the bronchoconstric­tor activity of methacholine, histamine and prostag­landin F2a (PGF2a) is considered to be mostly due to the direct activation of specific receptors on bronchial smooth muscle. In controlled studies, inhaled fruse­mide did not protect against methacholine in asthmatic patients [10, 23, 26], whereas, a reduction of sensitiv­ity was observed in two studies on normal subjects [27, 28]. Similarly, frusemide caused minimal [29], or no [24, 30], protection against histamine-induced bronchoconstriction, and has had no effect on the

bronchial response to PGF2t, [3 1[. ll did. however. cause s ignificam pro tection against thl! bronchocon­striction induced by leukotriene D~ (LTD4 ) [30].

The protective activity o r frusemide on the respi ra­tOry tract is not limited to bronchoconstriction. as it also protects agains t the tu ssive response to low chloride solutions [32. 33 ]. and to PGF~,. [3Jj. In contrast, it was ineffective on cough induced by citric acid [34j. capsaicin ]32), ami metabisulphite LJSj. Interesting ly. the bronchoconstrictor effect o f meta­bi!.ulphite is inhi bited by frusemide ]23. 24, 35j. but no t that o f PG F~u f3 I] , in contrast to the results obtaiued on cough induced by the same stimuli .

Possible mechanism of action

A number of Olher drugs affecting ion transpon have been investig<lled tO provide further information on the mechanism of ac t io n o f fru semi de in asthma. Bumeranide and piretanide are k1op diuretics or higher potency th~m fmsemide . as far as inhibition of Na?/K '/ Cl eo-transport and on mllriuretic activity are con­cerned 1361. They are also more pote nt than fruse­mide in inhibiting ion transport in the clog tracheal epithe lium 14 1, and electrically-induced conlracLion of guinea-pig bronchial su·ips in l'ilro [37] . However, when :tdministercd. by inhalation. to asthmat ic.: subjects at doses with equi vale nt diuretk activity. bumetanide provided significanlly lower protect-ion than frusemide agains t exerc ise- [38] and udenosine-i nduced f24j bronchoconstriction. and it had no effect against so­dium merabisulphite 124 1.

Unde r the same experimental cond itions, pirc tanide showed little protection againsl distilled wate r- IJ9l nnd metabisulphite-induccd bronchoconstriction [40]. Higher doses of pircwnide, however, caused signifi­cant. dose-related pr<>tection agaillst distilled water. and the drug was roughly equivalent t<> frusemide on an equimolar dose 139j. A newer loop diuretic, tora­semide, a lso had liule effect against distilled Wilier­induced bronchoconsrriction f41] . The reason for thilt dissociation between tl1e diuretic a nd the bronc ho­protective activities of loop diure Lics is not clear. The bronchoprotective activi ty may be mediated by a mechanism differe nt from the Na .. / K'/Cl cO-lransport inhibition. responsible for the diuretic acti vity. Alte r­natively, the kinetics of frusemide in the airways might be different from other diuretics. resulting in stronger or longer local activity. The phannacok inetic proper­tics of frusemide and orhe r loor diuretics after inha­lation have not been investigated. Their protective e ffect. however, does not appear to depend on lipid solubilit y, as bumetanide and torasemide are highly lipophilic, whereas frusemide and pirctanide have low lipid solubi lity [361.

lncre;~ :; ing infonnation is available on the bronchial effects of drugs active on differen t ion transport mechanisms. Inhalation of the Na'/K~ adenosine tri­phosphatase (ATPase) blocke r. ouabain. does not alter the bronchial responses to exerc ise Il l. or histamine

132 S. BlANCO ET AL.

(42], in asthmatic patients. The Na+ channeL blocker, amiloride, has been reported to slightly reduce bronchoconsniction, but not cough, induced by distilled water, and to have no significant effect on exercise, metabisulphite, methacholine and cold air-induced bronchoconstriction [43, 45]. The carbonic anhydrase inhibitor, acetazolamide, has been shown to reduce the bronchial response to cold air and metabisulphite [45]. Finally, it is interesting to note that sodium cro­moglycate, an anti-asthmatic drug with a spectrum of actjvities very similar to frusemide, has recently been shown to be a potent Cl· channel inhibitor in a mast cell derived tumour line [46].

These findings suggest that ion transport mechanisms participate in the control of bronchial reactivity in asthma, but they do not provide sufficient infonnation to detennine the exact mechanism(s) involved. As ion transport pathways are present in virtually every cell type, all cells suspected to be involved in the patho­genesis of bronchial hyperreactivity in asthma are po­tential targets for the action of frusemide. However, since frusemide has little protective activity on bron­choconstrictor stimuli acting directly on smooth muscle, an effect on these cells appears unlikely. The effects of loop diuretics [4, 5], and allergen challenge [ 47, 48], on ion transport in the airways have mostly been investigated on the tracheal epithelium of ex­perimental animals. Conflicting results have been re­ported on the effect of frusemide on human nasal epitheHum in vivo [23, 49]. The response of nasal epithelium, however, might not be representative of the action of frusemide at bronchial level, as we failed to observe consistent protection for nasal allergen chal­lenge following frusemide inhalation in patients with allergic rhinitis (unpublished results). Frusemide has been shown to inhibit allergen-induced histamine re­lease from rat peritoneal mast cells [50], and human blood cells [51], and to inhibit the release of histamine and leukotrienes from human lung fragments sensitized in vitro [52]. Other cells, which have been reported to be affected by frusemide, include guinea-pig eosino­phils [53], as well as human neutrophils [54, 55], al­veolar macrophages [56, 57], and bronchial epithelial cells [57]. No infonnation is available on the effect of loop diuretics on human airway neurotransmission. In the guinea-pig, frusemide and bumetanide inhibit both cholinergic and non-cholinergic neurally­mediated bronchoconstriction in vitro, through a mechanism not influenced by the presence of the epithelium, and which appears to be mediated by inhibition of Na+fK+fCl· eo-transport [37].

The possibility that the protective activity of fruse­mide on bronchial reactions is mediated by prostag­landins has been investigated using cyclooxygenase inhibitors. Oral treatment with indomethacin and flurbiprofen failed to affect the protective activity of frusemide on the response to distilled water and metabisulphite, respectively, [58, 59]. The effect of frusemide on exercise-induced bronchoconstriction was reversed by oral indomethacin treatment [60]. We have recently observed that inhalation of lysine

acetylsalicylate (LASA) strongly potentiates the protec­tive activity of frusemide against distilled water­induced responses [61], and that it has an additive ef­fect against a llergen-induced bronchoconstriction [21]. Inhaled sodium salicylate does not potentiate the ac­tivity of frusemide, suggesting that the effect of LASA is due to prostaglandin inhibition [62]. Surprisingly, the protective effect of frusemide on exercise-induced reactions also appears to be potentiated by inhaled LASA [63]. Thus, the effect of non-steroidal, anti­inflammatory agents on the protective activity of fruse­mide appears to depend on the route of administration and possibly on the stimuli and the drugs used.

Therapeutic perspectives

Taken together, these experimental studies indicate that frusemide has a wide spectrum of activity not confined to epithelial cells but extending to other cell types, including neutrophils, eosinophils, macrophages and nerve fibres, all of which are involved in the pathogenesis of asthma. These observations suggest that frusemide might provide a novel approach to the treatment of asthma, or might constitute a starting point for the development of new anti-asthma drugs. Frusemide has not caused bronchodilatation in any of the studies detailed above, or in a controlled study in which specific airway resistance and forced expiratory volume in one second were monitored for 5 h in a group of asthmatic patients with airway obstruction highly reversible after inhalation of fenoterol [64]. In a more recent study, frusemide did not interact with salbutamol-induced bronchodilatation, and the two drugs had an additive protective effect against distilled water-induced bronchoconstriction [65]. A beneficial effect of inhaled frusemide, however, can be expected in patients with bronchial hyperreactivity, due to the prevention of bronchoconstriction. This possibility is speculative at present, and controlled clinical studies to verify the hypothesis are currently being perfonned. Preliminary results from short-term clinical studies, showing a small beneficial effect of inhaled frusemide in patients with severe asthma [66-69], also need confirmation by ongoing clinical trials. Treatment with inhaled frusemide should, therefore, not be pre­scribed outside controlled studies until it has been formally validated.

The recent observation that the protective activity of frusemide is greatly potentiated by inhaled LASA and by other prostaglandin inhibitors [60, 62] raises the possibility that the association of the two drugs might provide increased therapeutic activity. Preliminary results from an open study indicate that combined treatment with inhaled frusemide and LASA had a significant steroid-sparing effect in a group of patients with severe, steroid-dependent asthma [70]. Appro­priate controlled studies are needed to confirm this observation, and to provide more information on the relative contribution of the two drugs. If confinned, these results might open new therapeutic perspectives, particularly for patients with severe asthma, for whom

INHALED LOOP DIURETICS IN ASTHMA 133

a satisfactory alternative to long-tenn therapy with sys­temic steroids is not currently available.

References

I. Bianco S , Robuschi M, Sestini P. t ' l al. - Osmolar­ity, bronchial reactivity and the protective effect of loop diuretics. In: Melillo G .. Normnn P.S .. Maronc G., eds. Respiratory Allergy. Toronto , BC Deckc r. 1990; pp. 119-130. 2. Al-Bazzaz FJ. - Regulation of salt and water trans­port across airway mucosa. Clin Chest Med, 1986; 7: 259-272. 3. Welsh MJ. - Mechanisms of airway epithelial ion transport. Clin Chest Med, 1986; 7: 273-293. 4. Widdicombe JH, Nathanson IT, Higland E. - Effect of "loop" diuretics on ion transport by dog tracheal epithe­lium. Am J Physiol, 1983; 245 (Cell Physiol 14): C388-C396. 5. Welsh MJ. - Inhibition of chloride secretion by furo­semide in canine tracheal epithelium. J Membr Bioi, 1983; 71: 219- 226. 6. Robuschi M , Gambaro G , Spagnotto S, Vaghi A, Bianco S. - Inhaled furosemide (F) is highly effective in preventing ultrasonically nebuJized water (UNH20) bronchoconstriction. Pulm Pharmacal, 1989; I: 187-191. 7. Bianco S, Vaghi A, Robuschi M. - Furosemide: a "new" antireactive agent. It works only by inhalation. Eur Respir J, 1988; 1: 2s. 8. Bianco S, Vaghi A, Robuschi M, Pasargiklian M. -Prevention of exercise-induced bronchoconstriction by inhaled frusemide. Lancet , 1988; ii: 252-255. 9. Robuschi M, Vaghi A , Gambaro G, Spagnotto S, Bianco S. Inhaled furosemide (F) is effective in preventing ultrasonically nc bulized 5.8(k NnCI bronchocon­s triction. Eur Re,rpir J, 1988: I (Suppl): 1 93~. 10. Grubbc RE. Hopp R, Oave NK , et nl. - Effect of lnhuled furosemide.: on the bronchial response to metha­choline and cold air hyperventilation challenges. J Allergy Clin lmmunol, 1990; 85: 881-884. 11. Magyar P, Debreczeni LA, Zsamboki G, Gyor Z. -Protective effect of inhaled furosemide on KCl-induced bronchospasm. Eur Respir ], 1991; 4: 605s. 12. Cogo A, Dal Negro R , Clini E, Turco P, Allegra L. - Protective effect of inhaled furosemide (F) against V A/Qc dis turbances induced by ultrasonically nebul i7.ed d istilled water (UNDW) in asthmatics. Eur Re.fpir .1, 199 1: 4 : 606s. 13. Moscato G. Oe llubinncu A. Fnhtgiani P. et (11. - In­haled furosemide prevents both the hronchoconstric tion and the increase in neutrophil c hemotactic ac tivity induced by ultrnsonk "fog'' of distilled wuter in as thmatics. A lii Nt' l' Respir Dis. 199 1; 143: 561 -566. 14. O'Bym c PM , Dolovich J, Hargreave FE. - Late asth­matic responses. Am Re1• f? t'SJlir Dis. 1987: 136: 74(}-75 1. 15. Bianco S, Pic roni M , Rcl1ni M. SLUrman A. Robuschi M. - Inhaled furosemide (F) prevents a llergen-induced bronchoconstriction in atopic asthmatics. Am Rev Respir Dis. 19!:18: 137 (Abstract}: 27. 16. Robuschi M , Picroni M, Refin i M, 1!1 of. - Preven­tion of antigen-induced early obstructive reaction by inhaled furosemide in (!IIC)pic) usthmatics und (ac tively sens it i7ed) guin~a-pigs. .r Afll!rgy Cl in 1111111111/tJI. 1990: 85: I 0-16. 17. Vcrdi:.Ull P, DiCarlo S. Baronti A, Binnco S. - Ef· feet of inhaled furosemide on the early response to antigen and subsequent change in airway reactivity in atopic pa­tients. Thorax, 1990; 45: 377-381.

18. Bianco S, Pieroni MG, Rcfini RM, et al. - Atten­uation of the bronchial response to allergen by inhal.ation of furosemide after allergen challenge. Am Rev Respir Dis, 1990; 141: A474. 19. Bianco S, Pieroni M, Refini M, Rottoli L, Sestini P. - Protective effect of inhaled furosemide on allergen­induced early and late asthmatic reactions. N Engl J Med, l989; 321: 1069-1073. 20. Sestini P, Vaghi A, Pieroni MG, er al. - Time course of the protccti ve effect of inhaled furosemide on UNH,O­induced bronchoconstrietion. Eur Respir J, 1991; 3: 237s. 21. Sestini P, Refini RM, Pieroni MG, et al . - Protec­tive effect of inhaled furosemide and lysine acetylsalicylate on a!Jergen-induced bronchial hyperreactivity. Am Rev Respir Dis, 1992; 145: A728. 22. Wooley MJ, Matsui S , Karlsson JA, O'Byrne PM. -Effect of inhaled furosemide on allergen-induced airway hyperresponsiveness and inflammation in dogs. Am Rev Respir Dis , 1990; 141: A363. 23. Nichol GN, Alton EWFW, Nix A, et al. - Effect of inhaled furosemide on metabisulfite- and methacholine­induced bronchoconstriction and nasal potential difference in asthmatic subjects. Am Rev Respir Dis, 1990; 142: 576-580. 24. O'Connor BJ, Chung KF, Chen-Wordell YM, Fuller RW, Bames PJ. - Effect of inhaled frusemide and bume­tanide on adenosine 5-monophosphate and metabisulphite­induced bronchoconstriction in asthmatics. Am Rev Respir Dis, 1991; 143: 1329-1334. 25. Polosa R, Leu LC, Holgate ST. - Inhibition of ad­enosine 5'-monophosphate- and methacholine-induced bronchoconstriction in asthma by inhaled furosemide. Eur Respir J, 1990; 3: 665-672. 26. Vaghi A, Robuschi M, Chilaris N, Bianco S. Inhaled furosemide (F) does not attenuate the bronchial response to methacholine (M) in asthmatics. Eur Respir J, 1988; I: 85s. 27. Polosa R, Rajakulasingan K, Gratziou C, Holgate ST. - Inhaled loop diuretics attenuate bronchial responsiveness to methacholine in normal subjects. Eur Respir J, 1991; 14: 605s. 28. Fujimura M , Sakamoto S, Kamio Y. - Effect of inhaled furosemide on bronchial responsiveness to metha­choline. N Engl .I Med, 1990; 322: 935- 936. 29. Vaghi A, Robuschi M, Berni F, Bianco S. - Effect of inhaled furosemide (F) on bronchial response to hista­mine (H) in asthmatics. Eur Respir J, 1988; 1: 406s. 30. Cheung 0, Bel EH, van der Veen H, Dijkman JH, Sterk PJ. - The effect of inhaled frusemide on histamine and leukotriene 0 4-induced airway narrowing in asthmatic subjects. Eur Respir J, 1990; 4: 605s. 31. Stone RA, Yeo TC, Bames PJ, Chung KF. - Fruse­mide inhibits cough but not bronchoconstriction to prostag­landin PGFz. in patients with asthma. Am Rev Respir Dis, 1991; 143: A548. 32. Ventresca PG, Nichol GM, Bames PJ, Chung KF. -Inhaled frusemide inhibits cough induced by low chloride solutions but not by capsaicin. Am Rev Respir Dis, 1990; 142: 143-146. 33. Stone RA, Bames PJ, Chung KF. - Frusemide and cough induced by low-chloride content solution: duration of action. Thorax. 1991; 46: 280P- 281P. 34. Robuschi M, Vaghi A, Gambaro G, et al. - Effect of inhaled furosemide on cough response to citric acid in normal subjects. Prog Resp Res, 1990; 24: 117-121. 35. O'Connor BJ, Chen-Worsdell YM, Ylmaz G, Barnes PJ, Chung KF. - Furosemide via a metered dose inhaled

134 S. BJANCO ET AL.

(MOl): dose-dependent inhibition of bronchoconstriction but not cough responses to sodium metabisulphite (MBS) in asthma. Am Rev Respir Dis, 1992; 145: A733. 36. Lant A. Diuretics: clinical pharmacology and therapeutical use (Part n. Drugs, 1985; 29: 57-87. 37. Elwood W, Lotvall JO, Bames P, Chung KF. - Loop diuretics inhibits cholinergic and noncholinergic nerves in guinea-pig. Am Rev Respir Dis, 1991; 143: 1345-1349. 38. Vaghi A, Robuschi M, Sestini P, et al. - Compari­son of inhaled frusemide and bumetanide in preventing ultrasonically nebulized water bronchoconstriction. Eur Respir J, 1990; 3: 93s. 39. Bianco S, Robuschi M, Vaghi A, et al. - Protective effect of inhaled piretanide on the bronchial obstructive re­sponse to ultrasonically ncbulized H,O. Am Rev Respir Dis, 1991; 143: A210. 40. Yeo CT, O'Connor BJ, Chen-Worsdell M, Barnes PJ, Chung KF. - Protective effect of loop diuretics, piretanide and frusemide, against mctabisulphite-induced bronchocon­striction in asthma. Eur Respir J, 1992; 5: 1184-1188. 41. Foresi A, Pelucchi A, Mastropasqua B, et al. Effect of inhaled furosemide and torasemide on bronchial obstructive response to ultrasonically nebulized distilled wa­ter (UNDW) in asthmatic subjects. Am Rev Respir Dis, 1992; 145: A374. 42. Knox AJ, Britton JR, Tattersfield AE. - Effect of sodium-transport inhibitors on bronchial reactivity in vivo. Clin Sci, 1990; 79: 325-330. 43. Wood AM, Lowry RH, Higenbottam TW. - Differ­ential effects of inhaled frusemide and amiloride on airway responsiveness to water and exercise in asthma. Am Rev Respir Dis, 1990; 141: A478. 44. Netzel MA, Hopp R, Buzzas R, et al. - Effect of inhaled amiloride on cold air (CAHC) and methacholine challenges (MC). Ann Allergy, 1991; 66: 82. 45. O'Connor BJ, Yeo CT, Chen-Worsdell YM, Bames PJ, Chung KF. - Airway responses to sodium metabisulphite are inhibited by inhaled acetazolamide but not by amiloride. Eur Respir J, 1991; 4: 377s. 46. Romanin Chr, Reinsprecht M, Pecht I, Schinder H. -The ami-allergic drug cromolyn is a potent blocker of Cl· channel activity in rat mucosal mast cells (RBL-2H3 line). Nayun Sch Arch Pharmacol, 1991; 343: 3'13. 47. Sestini P, Bienenstock J , Crowe SE, et al. - Ion transport in rat epithelium in vitro: role of capsaicin­sensitive nerves in allergic reactions. Am Rev Respir Dis, 1990; 141: 393-397. 48. Choi BW, Perdue M, Otis J, Manning PJ, O'Bytne PM. - The effects of allergen inhalation on ion transport in the tracheal epithelium in sensitized and control rats. Am Rev Respir Dis, 1992; 145: A368. 49. Wood AM, Dinh Xuan AT, Higenbottam TW, Cremona G, Lockart A. - Metoxamine and frusemide inhibit nasal transepithelial potential d ifference: a possible protective mechanism in the prevention of exercise-induced asthma. Am Rev Respir Dis, 1989; 139: A478. 50. Berti F, Rossoni G. Buschi A, Zuccari G, Villa LM. - Inhaled furosemide prevents immunological respiratory changes and mediator release in guinea-pig. Eur Respir J, 1991; 4: 445s. 51. Gato JJ, Croce M, Cosra-Manso E, Senz M, Oehling A. - Effect of furosemide on in vitro histamine release. Allergy, 1992; 47 (Suppl.): 141. 52. Anderson SD, Wei H, Temple DM. - Inhibition by furosemide of inflammatory mediators from lung fragments. N Engl J Med, 1991; 324: 131.

53. Perkins RA, Dent G, Chung KF, Bames PJ. - Effects of anion transport inhibitors and chloride ions on eosinophil respiratory burst activity. Am Rev Respir Dis, 1991; 143: A331. 54. Rottoli L, Rottoli P, Spamacci F, Bianco S. - In vitro effect of furosemide on 0 2 production. Eur Respir J, 1989; 2 (Suppl. 8): 861s. 55. Sestini P, Renzoni E, Vagliasindi M, Bianco S. Inhibitory effect of furosemide on FMLP-induced chemo­taxis of human granulocytes in vitro. Eur Respir J, 1990; 3: 348s. 56. Rottoli P, Rottoli L, Marino M, et al. - Effect of furosemide on lysosomal enzyme secretion by alveolar macrophages. Eur Respir J, 1990; 3: 307- 308s. 57. Scoloperto M, Marini M, Brasca C, Fasoli A, Mattoli S. - The protective effect of furosemide on the generation of superoxide anions by human bronchial. epithelial cells and pulmonary macrophages in vivo. Pulm Pharmacol, 1991; 4: 80-84. 58. Vaghi A, Berni F, Robuschi M, Sestini P, Bianco S. - Indomethacin (I) does not influence the protective effect of furosemide (F) against exercise-induced broncho­constriction. Eur Respir J, 2 (Suppl. 8): 790. 59. O'Connor BJ, Ridge SM, Barnes PJ, Chung KF. -The role of cyclooxygenase products in the inhibition of sodium metabisulphite-induced bronchoconstriction by furosemide in asthma. Am Rev Respir Dis, 1991; 143: A210. 60. Pavord ID, Wisniewski A, Tattersfield AE. - Inhaled frusemide and exercise-induced asthma: evidence of a role for inhibiting prostanoids. Am Rev Respir Dis, 1991; 143: A2LO. 61. Bianco S, Vaghi A, Pieroni MG, et al. - Potentiation of the protective effect of inhaled furosemide on the bron­chial obstructive response to ultrasonically nebulized water by inhaled lysine acetylsalicylate. Eur Respir J, 1991; 4: 606s. 62. Robuschi M, Vaghi A, Spagnotto S, et al. - Inhaled sodium salicylate does not enhance the protective activity of furosemide against ultrasonic fog. Am Rev Respir Dis, 1992; 145: A56. 63. Robuschi M, Scuri M, Vaghi A, et al. - Inhaled acetylsalicylic acid enl1ances the protective effect of furo­semide against exercise-induced bronchoconstriction. Am Rev Respir Dis, 1992; 145: A 729. 64. Sestini P, Pieroni MG, Refini RM, et al. - Effect of inhaled furosemide on respiratory function parameters of patients with reversible airway obstruction. In: Olivieri D., ed. New trends in pneumology and cardiology. Bologna, Monduzzi, 1990; pp. 55-59. 65. Vaghi A, Pieroni MG, Robuschi M. et al. - Preven­tive effect of fu rosemide and salbutamol on ultrasonically nebulized water. Eur Respir J, 1991; 4: 606s. 66. Chin T, Nussbaum E. Beneficial effect of aerosolized furosemide in pediatric asthma. Am Rev Respir Dis, 1991; 143: A624. 67. Nannini U, Pendino JC, Molfino NA, Slutsky AS. -Inhaled furosemide and salbutamol in acute asthma. Am Rev Respir Dis, 1992; 145: A422. 68. Hui KP, Koh TH, Tan WC. Effect of inhaled furosemide on persistent bronchoconstriction in patients with chronic asthma. Am Rev Respir Dis, 1992; 145: A736. 69. Brignani F. - Efficacy of inhaled furosemide in preventing bronchospasm. Allergy, 1992; 42 (Suppl.): 107. 70. Bianco S, Robuschi M, Vaghi A, er al. - Combined treatment with inhaled furosemide and lysine acetylsalicylate in steroid-dependent asthma. Eur Respir J, 1991; 4: 343-344s.