Rj73284_24_live 195.199

Hypertonic saline alters ion transport across the P.G. Middleton, K.A. Pollard, J.R. Wheatley Hypertonic saline alters ion transport across the human airway epithelium. P.G.
Middleton, K.A. Pollard, J.R. Wheatley. #ERS Journals Ltd 2001.
ABSTRACT: Aerosolized hypertonic saline is currently being investigated as a new agent for the treatment of impaired mucociliary clearance which occurs in many respiratory diseases. Mannitol aerosols, in particular dry powder inhalers, have been proposed as an alternative treatment to saline, offering the same osmotic load with other benefits. However, the effects of these hypertonic aerosols on airway epithelial ion transport processes have not been tested in human subjects in vivo. This report examines the effect of these solutions on airway ion transport using the nasal potential difference Seven healthy nonsmoking adult volunteers were studied. On different days, a dose- response curve was constructed for the saline added to Krebs N-[2-hydroxyethyl] piperazine-N9-[2-ethanesulphonic acid] (HEPES) diluent. The reversibility of this saline effect was measured, and the response to additional saline (500 mM) and mannitol(1 M) compared.
Hypertonic saline decreased nasal PD in a dose-related manner, with mean (SEM) decreases in PD (less negative) of 6.6 (1.5), 7.6 (1.6), 10.0 (2.0), 13.1 (2.9) and 14.8(3.2) mV (n~4) for addition of 150 mM, 250 mM, 500 mM, 1,200 mM and 2,000 mM NaCl to the Krebs HEPES diluent, respectively. The effect of hypertonic saline was fully reversible with washout for 3 min (presaline 15.9 (0.5) mV, postwashout 15.8 (1.1) mV, (n~4)). The hypertonic saline response was rapid in onset, sustained for at Fibrosis Association, the GovernmentEmployees Medical Research Fund and least 4 min, and decreased PD from 13.7 (1.7) mV to 5.1 (1.3) mV (n~7, pv0.001). In contrast, addition of mannitol to the perfusate did not significantly alter nasal PD, witha nonsignificant trend towards an increase (more negative) in the PD, (premannitol 13.9(1.6) mV, postmannitol 15.3 (2.0) mV, n~7).
As the osmotic stimulus of the 1 M mannitol is similar to that of the 500 mM sodium chloride, the divergent nasal potential difference responses suggest that the response tothe saline was specific to the sodium chloride itself and not the simultaneous change inosmolarity. This demonstrates that the human airway epithelium in vivo can respond totopical hypertonic saline independent of the altered osmolarity.
Eur Respir J 2001; 17: 195–199.
The human airways are covered by a thin layer of physical removal of the retained secretions or the use of fluid, the airway surface liquid (ASL), which protects pharmacological agents. Chest physiotherapy, an the underlying epithelial cells from drying, and traps example of physical removal of secretions, is extremely inhaled airborne particles and bacteria. The ASL is effective, but expensive and time consuming in the long thought to comprise a watery periciliary or sol layer, term. For this reason a number of pharmacological over which floats the gel or mucous layer. Though the therapies have been developed to increase MCC.
exact composition and volume of these 2 layers is Recently, interest has focussed on the use of currently debated, the ASL is thought to be largely nebulised therapy, in particular hypertonic saline and regulated by the ion transport processes across the mannitol, as potential new treatments to increase airway epithelium, namely sodium absorption and MCC. Topical application of hypertonic saline via chloride secretion. Optimal functioning of the ASL is nebuliser has been shown to increase MCC in patients required to maximise mucociliary clearance (MCC), with cystic fibrosis (CF) [1] and a 2 week clinical trial preventing accumulation of mucous and inhaled has demonstrated that 10 mL of 6% saline improved particles. Retained mucous and particles are a site for symptoms and lung function [2]. Similarly inhaled bacterial infection, which can then lead to airflow mannitol has been shown to increase MCC in subjects limitation, chronic cough and airway damage.
with CF [3] and idiopathic bronchiectasis [4]. However, As impaired MCC is thought to be involved in the the mechanism of action of both hypertonic saline and pathogenesis of a number of respiratory disorders, mannitol remains speculative. Whilst it has been treatments have been devised to improve MCC through hypothesised that the aerosols provide an osmotic load to the ASL, no studies have examined the effects of subjects had undergone nasal surgery, and all tests were hypertonic saline and mannitol on airway epithelial ion performed at least 4 weeks following an upper respiratory tract infection. The study was approved The nasal potential difference (PD) technique, which by the hospital ethics committee and all subjects gave involves passage of a small exploring electrode along the floor of the nose, allows in vivo measurement of iontransport across the human airway epithelium, bypass-ing the difficulties of in vitro techniques. The nasal PD technique has previously been used to study the effectof topical antibiotics on human airway epithelial ion The two-tailed paired t-test was used for comparison transport [5], and to investigate potential new aerosol and the null hypothesis was rejected at pv0.05. For therapies for CF [6]. In the current study, the effects of discussion purposes, increases and decreases refer to the topical application of hypertonic saline and mannitol absolute magnitude of the PD, which was lumen on nasal PD have been investigated, demonstrating that saline, but not mannitol, rapidly and reversiblydecreases nasal PD in normal human subjects.
Initial studies measured the response of the nasal epithelium to different concentrations of added NaCl.
Nasal PD was measured using previously described Following stabilisation of the PD in the Krebs HEPES methods [5, 7, 8]. Briefly, the exploring electrode diluent, the nose was perfused for 3 minutes with each consisted of a double lumen silicone rubber tube with of the following solutions: Krebsz150, Krebsz250, the openings of both lumens at the same site, 3 mm Krebsz500, Krebsz1,200 and Krebsz2,000 mM from the tip. One lumen was filled with an equal NaCl. This was associated with significant (p mixture of saline and electrocardiography electrode cream, connected to a high impedance voltmeter via a (1.6), 10.0 (2.0), 13.1 (2.9) and 14.8 (3.2) mV (n~4) silver/silver chloride electrode. The second lumen was (fig. 1). As the higher concentrations of saline present in perfused with the different solutions as outlined below, using a peristaltic pump which provided a continuous resulted in subjective discomfort in the nose during flow of 4 mL.min-1 throughout the perfusion period.
the testing, a solution of Krebsz500 mM NaCl was The reference electrode consisted of a second silver/ selected for further investigation. This gave approxi- silver chloride electrode placed over an area of abraded mately 70% of the maximal response without causing skin on the forearm, again connected to the voltmeter.
any discomfort. All subsequent studies of hypertonic Prior to recordings, the offset of the electrodes was saline in this paper were then performed with recorded values. In all cases, the tests started with aninitial period of stabilization using standard Krebs N-[2-hydroxyethyl]piperazine-N-[2-ethanesulphonic acid](HEPES) solution. Following stabilization of the nasal PD, the diluent was changed to the various hypertonicsolutions as indicated below, with different hypertonic responses measured on different days, in random order.
Perfusion commenced with Krebs HEPES solution of composition (mM): Naz 140, Kz 6, Ca2z 2, Mg2z 1, Cl- 152, glucose 10 and HEPES 10, titrated topH 7.4. Hypertonic solutions were produced volume- trically, so that the total concentrations of Naz and Cl- in the Krebsz500 mM saline were 640 mM and 652 mM, respectively. In the dose response testing, the solutions were perfused sequentially as indicated.
Due to the 3 mL dead space of the perfusion system, the new perfusate reached the catheter tip approxi- mately 45 s following solution change. Fresh stock solutions were prepared daily and diluted as required.
All solutions were perfused at room temperature Fig. 1. – The effect of additional saline on the nasal potentialdifference (PD) in four normal subjects. Each new solutionreached the nose approximately 45 s after the perfusate was changed, reflecting the dead space of the system. The solutionstested are indicated in the bar at the top of the graph. Data are Nonsmoking control subjects (4 female and 3 male, expressed as mean¡SEM. The PD is expressed in absolute terms,and was lumen negative. Arrows indicate KrebszNaCl solutions aged 18 – 40 yrs) with no history of respiratory disease of the following concentrations; A: 150 mM NaCl; B: 250 mM; were recruited from the hospital staff for testing. No nasal PD, with a trend to an increase in the PD, (2.0) mV, n~7, p~0.14)), as shown in figure 3.
This study has demonstrated that topical application of hypertonic saline, Krebsz500 mM NaCl, corre- sponding to a stimulus of y3% saline, decreases nasal potential difference. The response to the hypertonicsaline was rapid, reversible and dose related, suggestinga direct ion transport effect rather than nonspecific epithelial toxicity. In contrast, application of a similar osmotic load through hypertonic mannitol did not decrease the nasal PD, with a nonsignificant increase Fig. 2. – The effect of additional saline (500 mM) added to the (more negative). This suggests that the ionic composi- Krebs HEPES N-[2-hydroxyethyl]piperazine-N9-[2-ethanesulpho- tion of the saline, and not the osmotic load, was nic acid], on the nasal potential difference (PD) in four normal involved in the saline response. As amiloride pretreat- subjects. The new solution reached the nose approximately 45 s ment can block the saline response in normal subjects after the perfusate was changed (indicated by arrows), reflecting (unpublished data), passive diffusion of Cl- ions down the dead space of the system. After 1 min of perfusion with thehypertonic saline solution, the perfusate returned to Krebs the paracellular pathway would not explain the changes HEPES diluent to assess the effect of washout. Data are in nasal PD. Therefore it is hypothesized that the saline expressed as mean¡SEM. The PD is expressed in absolute terms, directly alters ion transport processes across the normal human airway epithelium. This is the first demonstra-tion in human subjects in vivo that increasing the NaClconcentration on the surface alters airway epithelial ion transport, independent of any effect of osmolarity.
Previous studies measuring the effect of hypertonic solutions on epithelial cells in vitro have found varying results. Early studies in canine trachea, a predominantly Cl- secreting epithelium, have shown that additionalluminal NaCl (75 mM) decreased short-circuit current (Isc) and increased tissue conductance [9]. The decreasein Isc predominantly reflected an increase in mucosal to serosal Cl- movement, which was proportional to the increased Cl- concentration on the mucosal surface.
Interestingly, addition of (150 mM) mannitol to theluminal surface decreased Naz absorption across the canine trachea without significant changes in either Cl- movement or tissue conductance. MAN et al. [10] demonstrated that addition of 100 mM mannitol to theluminal fluid decreased PD across the canine trachea by Fig. 3. – The effect of additional saline (500 mM) or mannitol 3.9 mV, with a similar decrease in Isc. Interestingly (1 M) added to the Krebs HEPES N-[2-hydroxyethyl]piperazine-N9-[2-ethanesulphonic acid], on the nasal potential difference there was a small increase in tissue resistance, opposite (PD) in 7 normal subjects. The new solution reached the nose to the increased conductance found by YANKASKAS et approximately 45 s after the perfusate was changed, reflecting the dead space of the system. Data are expressed as mean¡SEM.
In monolayers of cultured human nasal polyp cells, The PD is expressed in absolute terms, and was lumen negative.
& WILLUMSEN et al. [11] measured the effects of raised : saline; #: mannitol. ***: pv0.001 versus baseline presaline; luminal and serosal osmolality. Addition of 150 mMmannitol to the luminal surface was associated withdecreases (less negative) in both apical (Va) and To determine whether this response may be related to basolateral (Vb) membrane potentials, with small but epithelial toxicity, the reversibility of the hypertonic variable effects on the transepithelial membrane saline response was measured. As shown in fig. 2, potential (Vt) in different monolayers. Addition of following 1 min of perfusion with Krebsz500 mM 75 mM NaCl to the luminal solution also decreased NaCl, washout for 3 min reversed the response (presa- both Va and Vb, but did not alter Vt [11]. The line 15.9 (0.5) mV, postwashout 15.8 (1.1) mV, (n~4)).
nonsignificant increase in nasal PD found in the current In seven subjects the change to Krebsz500 mM study corresponds with the variable effects on Vt found NaCl was associated with a significant decrease in nasal in the nasal polyp monolayers; Va and Vb cannot be PD from 13.7 (1.7) mV to 5.1 (1.3) mV (pv0.001). This assessed using the nasal PD technique. Similarly, the response was rapid in onset and sustained for at least changes in epithelial cell volume noted in the nasal polyp cultures cannot be measured by the current (1,000 mM) to the perfusate did not significantly alter technique. Further studies will be necessary to deter- mine whether luminal mannitol does alter ion transport ESCHENBACHER et al. showed that hypertonicity, in vivo, with similar but opposite effects resulting in no irrespective of ionic composition, induced both bronch- oconstriction and cough [17]. Isosmotic saline solution However, the responses to luminal saline appear to induced neither bronchoconstriction or cough, but differ in the two studies with a significant decrease in isosmotic dextrose, with a low Cl- concentration, nasal PD in the current study contrasting with the lack induced cough in the absence of bronchoconstriction of change in Vt found by WILLUMSEN et al. [11]. This [17]. This suggests that hypertonicity and ionic may simply reflect the different doses used in the composition can, in certain situations, be stimuli for respective studies – the only dose reported in the different responses. The relationship between this and WILLUMSEN et al. study was 75 mM, whilst the smallest the nasal PD responses in the current study will require dose used in the current study was 150 mM. Another possible explanation for the differences between the two Finally, the differing nasal PD response to hyper- studies is the use of nasal polyp cells, which are known tonic saline and mannitol may have important to exhibit ion transport processes which differ from implications for the development of new treatments those found in normal airway epithelial cells [12]. The for lung diseases. Clinical trials will be necessary to nasal PD also avoids the difficulties of cell culture investigate each hypertonic solution individually, as the techniques, in particular the submersion culture process results from hypertonic saline and mannitol are not and the hormones and antibiotics required to maintain the cells in a viable state. Finally, another alternative In conclusion, this study has demonstrated that may be the presence of hormonal factors which may topical application of hypertonic saline induces a rapid control ion transport in vivo, but are not maintained and reversible decrease in nasal potential difference in when the cells are grown into monolayers in vitro.
vivo, most likely reflecting altered ion transport across Irrespective of the basis for the differences between the the human airway epithelium. The nonsignificant in vivo and the in vitro studies, the current report increase in nasal potential difference seen with provides important new information about the ion hypertonic mannitol suggests that the two solutions transport responses of the human airway epithelium in exert different effects on the epithelium, dependent on their composition. Further studies are necessary The nasal PD response to increasing concentrations to investigate the mechanisms involved in these of NaCl in the current study corresponds with the dose- related effects of saline on mucociliary clearance.
ROBINSON et al. [1] found that aerosols of 0.9%, 3%, 7% and 12% saline were associated with isotope to thank the subjects who took part in this clearance at 90 min of 13%, 20%, 24% and 26%, respectively. Whilst the MCC response may be related to the nasal PD effects demonstrated in the currentstudy, there are a number of other possibilities,including increased ciliary beat frequency in vivo [13]and direct effects on sputum [14]. Furthermore, thedivergent effects of hypertonic mannitol and saline on nasal PD do not correspond with the similar increasesin MCC in a variety of clinical situations [3, 4, 15].
Robinson M, Hemming AL, Regnis JA, et al. Effect of Hyperosmolarity is also a reliable stimulus to increasing doses of hypertonic saline on mucociliary provoke bronchoconstriction in susceptible subjects.
clearance in patients with cystic fibrosis. Thorax 1997; Hypertonic saline solutions have long been used in the laboratory as an indirect challenge agent, with more Eng PA, Morton J, Douglass JA, Riedler J, Wilson J, recent studies by ANDERSON et al. [16] studying the Robertson CF. Short-term efficacy of ultrasonically effects of inhaled mannitol. Whilst for the majority of nebulized hypertonic saline in cystic fibrosis. Pediatr subjects saline and mannitol produced qualitatively similar results, some subjects did show 10-fold higher Robinson M, Daviskas E, Eberl S, et al. The effect of inhaled mannitol on bronchial mucus clearance in et al. [17] compared the effects of different solutions cystic fibrosis patients: a pilot study. Eur Respir J 1999; with the same osmolarity on the airways of asthmatic subjects. They demonstrated that 4% saline solution Daviskas E, Anderson SD, Eberl S, Chan H-K,Bautovich G. Inhalation of dry powder mannitol was significantly more potent at inducing bronchocon- striction than 18.3% dextrose in 1% saline, though both bronchiectasis. Am J Respir Crit Care Med 1999; solutions had the same osmolarity. Similarly MAGYAR et al. [18] has reported that 1.3 M KCl was more potent than 1.7 M NaCl, (both solutions prepared at 10% weight/volume). This suggests that as well as the epithelial sodium absorption. Am J Respir Crit Care osmotic stimulus, the ionic composition may also be involved in bronchoconstrictor responses.
Smith SN, Middleton PG, Chadwick S, et al. The in It is well recognized that both the osmolarity and vivo effects of milrinone on the airways of cystic ionic composition of an aerosol are important in the fibrosis mice and human subjects. Am J Respir Cell induction of cough. In an elegant series of experiments, Middleton PG, Geddes DM, Alton EWFW. Effect of Wong LB, Miller IF, Yeates DB. Pathways of amiloride and saline on nasal mucociliary clearance substance P stimulation of canine tracheal ciliary and potential difference in cystic fibrosis and normal beat frequency. J Appl Physiol 1991; 70: 267 – 273.
subjects. Thorax 1993; 48: 812 – 816.
Wills PJ, Hall RL, Chan W, Cole PJ. Sodium chloride Middleton PG, Caplen NJ, Gao X, et al. Nasal increases the ciliary transportability of cystic fibrosis application of the cationic liposome DC-Chol:DOPE and bronchiectasis sputum on the mucus-depleted does not alter ion transport, lung function or bacterial bovine trachea. J Clin Invest 1997; 99: 9 – 13.
growth. Eur Respir J 1994; 7: 442 – 445.
Daviskas E, Anderson SD, Gonda I, et al. Inhalation Yankaskas JR, Gatzy JT, Boucher RC. Effects of hypertonic saline aerosol enhances mucociliary of raised osmolarity on canine tracheal epithelial ion clearance in asthmatic and healthy subjects. Eur Respir transport function. J Appl Physiol 1987; 62: 2241 – Anderson SD, Brannon JD, Spring J, et al. A new Man SFP, Hulbert W, Park DSK, Thomson ABR, method for bronchial-provocation testing in asthmatic Hogg JC. Asymmetry of canine tracheal epithelium: subjects using a dry powder mannitol. Am J Respir osmotically induced changes. J Appl Physiol 1984; 57: Crit Care Med 1997; 156: 758 – 765.
Willumsen NJ, Davis CW, Boucher RC. Selective Alteration in osmolarity of inhaled aerosols cause response of human airway epithelia to luminal but not bronchoconstriction and cough, but absence of a serosal solution hypertonicity. Possible role for permeant anion causes cough alone. Am Rev Respir transducer. J Clin Invest 1994; 94: 779 – 787.
Magyar P, Dervaderics M, To´th A. Bronchial Bernstein JM, Yankaskas JR. Increased ion transport challenge with hypertonic KCl solution in the Otolaryngol Head Neck Surg 1994; 120: 993 – 996.

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