Clin. Otolaryngol. 1992, 17, 351-353
The effects of diuretics on nasal transmucosal potential difference ELEANOR J.STEWART,* MICHAEL J.CINNAMOND,* PAUL NICHOLLSt & C.FRED STANFORDt Eve and Ear Clinic, *Royal Victoriu Hospital, Grosvenor Road, Belfhsr BTl2 6BJ, and tDeparlment of Respiratory Medicine, Royal Vicroria Hospital, Belfast BT12 6BJ, Northern Ireland Accepted for publication 7 April 1992 STEWART E . J . , C I N N A M O N D M . J . , NICHOLLS P . & S T A N F O R D C . F .
(1992) Clin. Otolaryngol. 17, 351 -353
The effects of diuretics on nasal transmucosal potential difference The effects of application of frusemide, amiloride and bumetanide on nasal transmucosal potential difference (NTPD), at rest and during exercise, were studied in 8 normal subjects. In a double-blind placebo controlled study, 8 volunteers had NTPD recorded at 4-min intervals during 12-min periods of rest. before and after treatment, during 12 min of exercise, and recovery. Application of placebo, frusemide and bumetanide did not significantly alter NTPD at rest. Amiloride caused a significant reduction (P < 0.025). During exercise there was a significant rise in NTPD with placebo (P < 0.05), frusemide (P< 0.05) and amiloride (P < 0.05). There was no increase in NTPD during exercise with bumetanide. Keywords nasal ~ransrnucosalpotentialdiflerence
Cells generate the transmembrane potential diffcrence which reflects the total electromotive forces of active and passive ion transport systems. In the respiratory mucosa, ion transport may be implicated in the production and clearance of secretions.' Measurement of nasal transmucosal potential difference (NTPD) as a diagnostic tool has been investigated in cystic fibrosis. respiratory discascs of the newborn, and in adult chest It is recognized that patients with cystic fibrosis have a very high lumen negative potential difference and that this may be related to an inability to absorb chloride ions.' It has also been shown that this high potcntial difference and that of normal subjects can be reduccd by the application of amiloride. a sodium channel blocker.'," In view of the recognized effects of the diuretics amiloride, frusemide and bumetanide on cells. we have studied the effect of their application upon exercise induced potential difference changes in normal nas''1 1 mucosa.
Materials and methods Eight male volunteers aged between 20 and 30 (mean age 24) were recruited. They had no history of nasal or respiratory Correspondence: E.J.Stewart. Wards 1 and 2. Royal Victoria Hospital, Grosvenor Road. Belfast BT12 6BJ, Northern Ireland
fritsemide
amiloride
buametanide
exercise
disease and had been free from upper respiratory tract infections for at least 2 weeks. All were non-smokers and were not taking any medication. Nasal transmucosal potential difference (NTPD) measurements were made using a modification of a previously described method.6 The reference bridge was a calomel electrode placed over a 3-mm diameter area of lightly abraded skin on the forearm. The exploring bridge was a silastic Foley catheter size 8 (Simpla), filled with a mixture of Ringer's solution and electrode cream. NTPD readings were taken by advancing the tip of the catheter along the floor of the nose as described in a previous study.' At the maximum recorded voltage, usually reached at 3 cm, the catheter was marked; recordings were taken from this site throughout the study. The bridges were connected to a high impedance voltmeter. Readings were averaged over 5 s by a portable computer. NTPD readings were taken at 4-min intervals during 12 min of rest pre and post-treatment, during 12 min of exercise. and recovery. Each of the 8 volunteers received medication in a doubleblind fashion, randomized by a latin square design. All medications were applied using a pump action atomirer, in a total volume of 0.24 ml per nostril. They were: normal saline (placebo), frusemide 1.0 mg, bumetanide 25.0 pg, or amiloride 250pg. Exercise was performed on a cycle
351
352 E.J.Stewart e l al.
Table 1. The change in nasal transmucosal potential difference (NTPD) at rest and after 12 min exercise, on local application of placebo. frusemide, amiloride and bumetanide (mV (s.e.m.))
Drug
Resting
Post-treatment
Peak exercise
Placebo
14.0
(0.75)
13.9
(0.87)
20.25 (2.4)
Frusemide
13.3
(1.6)
13.8
(1.76)
19.3
Amiloride
13.96 (1.14)
10.9
(0.52)
14.25 (1.46
Bumetanide 11.84 (1.24)
13.85 (1.8)
(2.3)
14.76 (2.0)
ergometer. each volunteer attaining a pulse rate of 80% maximum predicted for their age and sex. The tests were performed at the same time of day. The study was approved by The Research Ethical Committee, Faculty of Medicine, The Queens University of Belfast. Statistics were calculated using Student's paired t-test.
Results The results are shown in Table 1. It can be seen that the application of saline did not significantly alter resting nasal transmucosal potential difference ( P > 0.5), but there was a significant rise on exercise (P< 0.05). There was no significant change in NTPD within 12 niin of application of frusemide. The combination of frusemide and exercise led to a significant increase ( P .< 0.05). Amiloride significantly reduced NTPD at rest ( P < 0.025). There was a significant rise during exercise compared with the resting post-treatment value ( P < 0.05), but not with the resting pretreatment value (P> 0.5). Thc peak exercise value was significantly less than the peak exercise value achieved with placebo (P < 0.05). The application of bumetanide did not alter resting NTPD and there was no significant rise during exercise (P> 0. I).
Discussion These results confirm previous work showing that saline applied to the nasal mucosa does not alter the NTPD and that exercise does increase this v a l u ~ .We ~ ~ have ~ also confirmed the observation that locally applied amiloride reduces the NTPD in mucosa.6 It has been postulated that, at rest, sodium ions are absorbed followed by chloride ions. This results in the normal negative luminal potential. Block of the sodium channels by amiloride should make the lumen relatively more positive.6 During exercise, secretion is the dominant factor rather than absorption, since this is required to humidify the inspired air.' This secretion may be due to
chloride entry across the chloride channels, followed by sodium via the paracellular route.'" The current work shows that an increase in negative potential is still possible during exercise despite block of the cellular sodium channels by amiloride. Both frusemide and bunietanidc are used clinically as loop diuretics." It is interesting to note, however, that when these diuretics are inhaled they have different effects upon the response to various stimuli in asthmatic subjects. Thus frusemide but not bumetanide prevents asthmatics from developing exercise,'' antigen'' and metabisulphite" induced asthma. Both diuretics are known to work on the Na'/ZCl-/ K' co-transport exchange mechanism at the basolateral membrane." Frusemide action is influenced to some degree by prostaglandin^,'^ and its activity can be reduced by the use of non-steroidal anti-inflammatory drugs. Humetanide is the more effective as a diuretic on a molar basis. It has been shown that applied to the outside surface of frog skin it reduces the membrane resistance while tiuseniide increases it." It is also a much more effective inhibitor of chloride transport in ascites tuniour cells than frusemide." The current work demonstrates that while frusemide does not alter the normal exercise induced increase in NTPD, bumetanide suppresses it. This may be related to its effect on chloride channels.
References KILBURN K.H. (1968) A hypothesis for pulmonary clearance and its implications. Am. Rev. Resp. Dis. 98, 449-463 KNOWLES M.R., GATZY J.T. & BOUCHERR.C. (1981) Increased bioelectric potential difference across respiratory epithelia in cystic fibrosis. N . Engl. J . Med. 305, 1489-1495 GOWAN C.W., LAWSON E.E., GINGRAS J.. ROUCHEH R.C., GATZY J.T. & KNOWLES M.R. (1988) Electric potential difference and ion transport across nasal epithelium of term neonates: Correlation with mode of delivery. transient tachypnoea of the newborn, and respiratory rate. J . Paediurrics 113, 121-127 ALTON E.W.. HAYJ.G.. MIJNROC . & GEDDES D.M. (1987) Measurement of nasal potential difference in adult cystic fibrosis, Young's syndrome and bronchiectasis. Thorc~u42, 8 15-8 I 7 STASFORD C.F., ALUSWAIDAN S., Lr WANPo A,, MORRISON G.. REDMONDA,, DODGEJ.A., NlCHOLLS D.P., STEWART E.J. & MCELNAY J . (1992) The influence of exercise upon the nasal transmucosal potential difference in healthy subjects and patients with cystic fibrosis. Thorax (in press) KNOWLESM.R., CARSONJ.L., COLLIER J.L., GATZYJ.T. & BOUCHER R.C. (1981) Measurements of nasal transepithelial electric potential differences in normal human subjects in vivo. Am. Rev. Resp. Dis. 124, 484-490 HARRIS W., GIEBALY K.. ALUSWAIIIAN S . & STANFORD C.F. (1990) Alteration in nasal transepithelial potential difference during exercise. Oroluryngol. Hrad Neck Stirg. 103, 194 (abstr.) E.J., CINNAMOND M.J., NICHOILSP. & STANFORD C.F. STEWART (1992) The effect of methacholine o n nasal transepithelial potential difference. Clin. 0toluryigolog.v 17, 93 (abstr.) R.L. (1983) Exercise induced STANFORD C.F. & STANFORD rhinorrhoea (Athlete's nose). Br. Med. J . 21, 60
Eflects of diuretics on nasal transmucosal potential drflerence 353
10 TURNBEKG L. (1991) Cellular basis of diarrhoea. J . RUT. Coll. Phj.siriatts 2 5 , 53-62 11 FEICP.U. (1986) Cellular mechanisms of action of loop diuretics
implications for drug effectiveness and adverse effects. Am. J . Curiliol. 57, 14A-19A 12 Woou A . M . , LOWRYR.H. & HIGESBOTTAM (1990) Effect of inhaled diuretics on airway challenge in asthma. Eurupcnn Respir. J . 3 , 124s (abstr.) 13 BIANO S.. REFINI R.M., PIERONI M.G.. FERRETTI B.. SESTINI P. & VAGLIASINDI M. (1991) Potentiation of the protective effect of inhaled frusemide on allergen-induced bronchoconstriction by inhaled lysine acetylsalicylate. Eurup. Respir. J . 4 (Suppl. 14), 235s (abstr.) 14 YEO C.T.. O'CONNORR.J.. CHEN-WORSDELL M., BARNESP.J. &
CHU~SGK.F. (1991) Protective effect of loop diuretics, piretamide and frusemide, against nietabisuphite induced bronchoconstriction in asthma. Eurup. Rcspir. J . 4 (Suppl. 14), 378 (abstr.) 15 ACKERG . M . , MOLIMARD M . , REGNARDJ.. N A L ~ NE.. E FRECHE CH.,MARSACJ . & LOCKHART A. (1991) Frusemide inhibits PGE2 synthesis by cultured cell in human nasal and bronchial epithelia. Europ. Respir. J . 4 (Suppl. 14), 347s (abstr.) 16 KRAMERH.J. (1976) Effects of bumetanide on sodium transport of the isolated frog skin and on renal Na K ATPase. Pliarmarolugy 14, 48 1-489 17 CANDIA O.A. & SCHOEN H.F. (1978) Selective effects of bumetanide on chloride transport in bullfrog cornea. Am. J . Physiol. 234, 297-301
The effects of diuretics on nasal transmucosal potential difference ELEANOR J.STEWART,* MICHAEL J.CINNAMOND,* PAUL NICHOLLSt & C.FRED STANFORDt Eve and Ear Clinic, *Royal Victoriu Hospital, Grosvenor Road, Belfhsr BTl2 6BJ, and tDeparlment of Respiratory Medicine, Royal Vicroria Hospital, Belfast BT12 6BJ, Northern Ireland Accepted for publication 7 April 1992 STEWART E . J . , C I N N A M O N D M . J . , NICHOLLS P . & S T A N F O R D C . F .
(1992) Clin. Otolaryngol. 17, 351 -353
The effects of diuretics on nasal transmucosal potential difference The effects of application of frusemide, amiloride and bumetanide on nasal transmucosal potential difference (NTPD), at rest and during exercise, were studied in 8 normal subjects. In a double-blind placebo controlled study, 8 volunteers had NTPD recorded at 4-min intervals during 12-min periods of rest. before and after treatment, during 12 min of exercise, and recovery. Application of placebo, frusemide and bumetanide did not significantly alter NTPD at rest. Amiloride caused a significant reduction (P < 0.025). During exercise there was a significant rise in NTPD with placebo (P < 0.05), frusemide (P< 0.05) and amiloride (P < 0.05). There was no increase in NTPD during exercise with bumetanide. Keywords nasal ~ransrnucosalpotentialdiflerence
Cells generate the transmembrane potential diffcrence which reflects the total electromotive forces of active and passive ion transport systems. In the respiratory mucosa, ion transport may be implicated in the production and clearance of secretions.' Measurement of nasal transmucosal potential difference (NTPD) as a diagnostic tool has been investigated in cystic fibrosis. respiratory discascs of the newborn, and in adult chest It is recognized that patients with cystic fibrosis have a very high lumen negative potential difference and that this may be related to an inability to absorb chloride ions.' It has also been shown that this high potcntial difference and that of normal subjects can be reduccd by the application of amiloride. a sodium channel blocker.'," In view of the recognized effects of the diuretics amiloride, frusemide and bumetanide on cells. we have studied the effect of their application upon exercise induced potential difference changes in normal nas''1 1 mucosa.
Materials and methods Eight male volunteers aged between 20 and 30 (mean age 24) were recruited. They had no history of nasal or respiratory Correspondence: E.J.Stewart. Wards 1 and 2. Royal Victoria Hospital, Grosvenor Road. Belfast BT12 6BJ, Northern Ireland
fritsemide
amiloride
buametanide
exercise
disease and had been free from upper respiratory tract infections for at least 2 weeks. All were non-smokers and were not taking any medication. Nasal transmucosal potential difference (NTPD) measurements were made using a modification of a previously described method.6 The reference bridge was a calomel electrode placed over a 3-mm diameter area of lightly abraded skin on the forearm. The exploring bridge was a silastic Foley catheter size 8 (Simpla), filled with a mixture of Ringer's solution and electrode cream. NTPD readings were taken by advancing the tip of the catheter along the floor of the nose as described in a previous study.' At the maximum recorded voltage, usually reached at 3 cm, the catheter was marked; recordings were taken from this site throughout the study. The bridges were connected to a high impedance voltmeter. Readings were averaged over 5 s by a portable computer. NTPD readings were taken at 4-min intervals during 12 min of rest pre and post-treatment, during 12 min of exercise. and recovery. Each of the 8 volunteers received medication in a doubleblind fashion, randomized by a latin square design. All medications were applied using a pump action atomirer, in a total volume of 0.24 ml per nostril. They were: normal saline (placebo), frusemide 1.0 mg, bumetanide 25.0 pg, or amiloride 250pg. Exercise was performed on a cycle
351
352 E.J.Stewart e l al.
Table 1. The change in nasal transmucosal potential difference (NTPD) at rest and after 12 min exercise, on local application of placebo. frusemide, amiloride and bumetanide (mV (s.e.m.))
Drug
Resting
Post-treatment
Peak exercise
Placebo
14.0
(0.75)
13.9
(0.87)
20.25 (2.4)
Frusemide
13.3
(1.6)
13.8
(1.76)
19.3
Amiloride
13.96 (1.14)
10.9
(0.52)
14.25 (1.46
Bumetanide 11.84 (1.24)
13.85 (1.8)
(2.3)
14.76 (2.0)
ergometer. each volunteer attaining a pulse rate of 80% maximum predicted for their age and sex. The tests were performed at the same time of day. The study was approved by The Research Ethical Committee, Faculty of Medicine, The Queens University of Belfast. Statistics were calculated using Student's paired t-test.
Results The results are shown in Table 1. It can be seen that the application of saline did not significantly alter resting nasal transmucosal potential difference ( P > 0.5), but there was a significant rise on exercise (P< 0.05). There was no significant change in NTPD within 12 niin of application of frusemide. The combination of frusemide and exercise led to a significant increase ( P .< 0.05). Amiloride significantly reduced NTPD at rest ( P < 0.025). There was a significant rise during exercise compared with the resting post-treatment value ( P < 0.05), but not with the resting pretreatment value (P> 0.5). Thc peak exercise value was significantly less than the peak exercise value achieved with placebo (P < 0.05). The application of bumetanide did not alter resting NTPD and there was no significant rise during exercise (P> 0. I).
Discussion These results confirm previous work showing that saline applied to the nasal mucosa does not alter the NTPD and that exercise does increase this v a l u ~ .We ~ ~ have ~ also confirmed the observation that locally applied amiloride reduces the NTPD in mucosa.6 It has been postulated that, at rest, sodium ions are absorbed followed by chloride ions. This results in the normal negative luminal potential. Block of the sodium channels by amiloride should make the lumen relatively more positive.6 During exercise, secretion is the dominant factor rather than absorption, since this is required to humidify the inspired air.' This secretion may be due to
chloride entry across the chloride channels, followed by sodium via the paracellular route.'" The current work shows that an increase in negative potential is still possible during exercise despite block of the cellular sodium channels by amiloride. Both frusemide and bunietanidc are used clinically as loop diuretics." It is interesting to note, however, that when these diuretics are inhaled they have different effects upon the response to various stimuli in asthmatic subjects. Thus frusemide but not bumetanide prevents asthmatics from developing exercise,'' antigen'' and metabisulphite" induced asthma. Both diuretics are known to work on the Na'/ZCl-/ K' co-transport exchange mechanism at the basolateral membrane." Frusemide action is influenced to some degree by prostaglandin^,'^ and its activity can be reduced by the use of non-steroidal anti-inflammatory drugs. Humetanide is the more effective as a diuretic on a molar basis. It has been shown that applied to the outside surface of frog skin it reduces the membrane resistance while tiuseniide increases it." It is also a much more effective inhibitor of chloride transport in ascites tuniour cells than frusemide." The current work demonstrates that while frusemide does not alter the normal exercise induced increase in NTPD, bumetanide suppresses it. This may be related to its effect on chloride channels.
References KILBURN K.H. (1968) A hypothesis for pulmonary clearance and its implications. Am. Rev. Resp. Dis. 98, 449-463 KNOWLES M.R., GATZY J.T. & BOUCHERR.C. (1981) Increased bioelectric potential difference across respiratory epithelia in cystic fibrosis. N . Engl. J . Med. 305, 1489-1495 GOWAN C.W., LAWSON E.E., GINGRAS J.. ROUCHEH R.C., GATZY J.T. & KNOWLES M.R. (1988) Electric potential difference and ion transport across nasal epithelium of term neonates: Correlation with mode of delivery. transient tachypnoea of the newborn, and respiratory rate. J . Paediurrics 113, 121-127 ALTON E.W.. HAYJ.G.. MIJNROC . & GEDDES D.M. (1987) Measurement of nasal potential difference in adult cystic fibrosis, Young's syndrome and bronchiectasis. Thorc~u42, 8 15-8 I 7 STASFORD C.F., ALUSWAIDAN S., Lr WANPo A,, MORRISON G.. REDMONDA,, DODGEJ.A., NlCHOLLS D.P., STEWART E.J. & MCELNAY J . (1992) The influence of exercise upon the nasal transmucosal potential difference in healthy subjects and patients with cystic fibrosis. Thorax (in press) KNOWLESM.R., CARSONJ.L., COLLIER J.L., GATZYJ.T. & BOUCHER R.C. (1981) Measurements of nasal transepithelial electric potential differences in normal human subjects in vivo. Am. Rev. Resp. Dis. 124, 484-490 HARRIS W., GIEBALY K.. ALUSWAIIIAN S . & STANFORD C.F. (1990) Alteration in nasal transepithelial potential difference during exercise. Oroluryngol. Hrad Neck Stirg. 103, 194 (abstr.) E.J., CINNAMOND M.J., NICHOILSP. & STANFORD C.F. STEWART (1992) The effect of methacholine o n nasal transepithelial potential difference. Clin. 0toluryigolog.v 17, 93 (abstr.) R.L. (1983) Exercise induced STANFORD C.F. & STANFORD rhinorrhoea (Athlete's nose). Br. Med. J . 21, 60
Eflects of diuretics on nasal transmucosal potential drflerence 353
10 TURNBEKG L. (1991) Cellular basis of diarrhoea. J . RUT. Coll. Phj.siriatts 2 5 , 53-62 11 FEICP.U. (1986) Cellular mechanisms of action of loop diuretics
implications for drug effectiveness and adverse effects. Am. J . Curiliol. 57, 14A-19A 12 Woou A . M . , LOWRYR.H. & HIGESBOTTAM (1990) Effect of inhaled diuretics on airway challenge in asthma. Eurupcnn Respir. J . 3 , 124s (abstr.) 13 BIANO S.. REFINI R.M., PIERONI M.G.. FERRETTI B.. SESTINI P. & VAGLIASINDI M. (1991) Potentiation of the protective effect of inhaled frusemide on allergen-induced bronchoconstriction by inhaled lysine acetylsalicylate. Eurup. Respir. J . 4 (Suppl. 14), 235s (abstr.) 14 YEO C.T.. O'CONNORR.J.. CHEN-WORSDELL M., BARNESP.J. &
CHU~SGK.F. (1991) Protective effect of loop diuretics, piretamide and frusemide, against nietabisuphite induced bronchoconstriction in asthma. Eurup. Rcspir. J . 4 (Suppl. 14), 378 (abstr.) 15 ACKERG . M . , MOLIMARD M . , REGNARDJ.. N A L ~ NE.. E FRECHE CH.,MARSACJ . & LOCKHART A. (1991) Frusemide inhibits PGE2 synthesis by cultured cell in human nasal and bronchial epithelia. Europ. Respir. J . 4 (Suppl. 14), 347s (abstr.) 16 KRAMERH.J. (1976) Effects of bumetanide on sodium transport of the isolated frog skin and on renal Na K ATPase. Pliarmarolugy 14, 48 1-489 17 CANDIA O.A. & SCHOEN H.F. (1978) Selective effects of bumetanide on chloride transport in bullfrog cornea. Am. J . Physiol. 234, 297-301
