Respiratory Physiology & Neurobiology 131 (2002) 245– 253 www.elsevier.com/locate/resphysiol
Inhaled sodium fluoride decreases airway responsiveness to acetylcholine analogs in vivo Weimin Zhao b,1, Sonia Rouatbi a, Zouhair Tabka a, Herve´ Gue´nard b,* b
a Laboratoire de Physiologie, Faculte´ de Me´decine de Sousse, Sousse, Tunisia Laboratoire de Physiologie, Faculte´ Victor Pachon, Uni6ersite´ de Bordeaux 2, 146 rue Leo Saignat, 33076 Bordeaux Cedex, France
Accepted 30 April 2002
Abstract The study was conducted to characterize the action of NaF, which had relaxing property in carbachol precontracted isolated bovine bronchus, on airway responsiveness challenged by acetylcholine receptor agonists in rats and asthmatic humans.Tracheal flow rate and airway resistance were measured in anaesthetized rats. NaF was delivered either before carbachol challenge or together with carbachol. Patients with mild asthma were challenged with methacholine aerosol, and NaF was delivered when FEV1 fell by more than 20%. The results indicated that: (1) in rats NaF significantly inhibited carbachol-induced bronchial constriction when inhaled prior to carbachol challenge as airway resistances in the NaF and NaF + verapamil groups were significantly lower than those in the control group; (2) NaF significantly reversed carbachol or methacholine-induced bronchial constriction in asthmatic patients. In conclusion, NaF, delivered in form of aerosol, reduced bronchial responsiveness to carbachol in rats and had a bronchodilating effect on rat and human airways precontracted by inhalation of acetylcholine analogs. © 2002 Elsevier Science B.V. All rights reserved. Keywords: Airways, responsiveness, resistance; Chemical agents, NaF; Disease, asthma; Hyperreactivity, bronchial; Mammals, humans; Mammals, rat; Smooth muscle, airway
1. Introduction A previous in vitro study (Zhao and Gue´nard, 1997) suggested that sodium fluoride (NaF) had * Corresponding author. Tel.: +33-557-571-360; fax: + 33557-571-501. E-mail address:
[email protected] (H. Gue´nard). 1 Present adress: Department of Physiology, University of Saskatchewan, Saskatoon, Canada.
either contracting or relaxing effects on bovine bronchial smooth muscle. Its contractile effect was possibly related to the modulation of calcium channels via a PKC-dependent pathway (Ratz and Blackmore, 1990; Adeagbo and Triggle, 1991), whereas the relaxing effect was attributed to the inhibitory action of NaF on glycolysis (Zhao and Gue´nard, 1997). Fluoride is an inhibitor of enolase, an enzyme in the glycolysis pathway leading to phosphoenolpyruvate (Mayes, 1987). The relaxing effect was attributed to this
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metabolic effect as, in vitro, the addition of pyruvate to the solution reversed the NaF-induced relaxation on carbachol-precontracted bronchial segments (Zhao and Gue´ nard, 1997). The contracting effect of NaF was inhibited in the presence of the verapamil, a voltage-dependent calcium channel inhibitor. The present study was designed to provide further information on these actions in vivo in rats as well as in asthmatic patients. In the first series of experiments, we investigated the combined effect of NaF and verapamil on rat airway responsiveness against carbachol challenge. In the light of the results of the animal experiments, we applied a similar experimental protocol to human. To optimize the local action and minimize their general adverse effects, the drugs mentioned above including the NaF and verapamil solutions were delivered directly to the bronchi by aerosol during the challenge.
2. Materials and methods
2.1. Protocol 1: NaF applied prior to carbachol challenge in rats Fifty adult male Wistar rats were assigned randomly into five groups: one group received an aerosol of isotonic saline as control, while the others were treated with one of the following drugs: verapamil (0.01 M), NaF (0.5 M), verapamil (0.01 M) with NaF (1 M) or verapamil (0.01 M) with NaF (0.5 M). Animals were subsequently tested for carbachol responsiveness. Animal experiment protocols used in the present study were approved by the committee on experimental animal care and supply of the Universties of Bordeaux and were in agreement with European Directives (86/609/CEE). The rats were anesthetized intraperitoneally with ketamine (150 mg/kg). After dissecting the neck, the trachea was exposed by a mid-line incision and a tracheal cannula inserted. A second balloon tipped catheter was inserted into the lower 1/3 of the esophagus and connected to a pressure transducer to measure the intra-esophageal pressure (Peso). A small pneumotachograph (PTG 8431B, Hans
Rudolph, Kansas, USA) was connected, when needed, to the tracheal cannula to measure flow rate. The total dead space of the PTG and the cannula outside the trachea was about 0.4 ml. To avoid change in ventilation while the rat breathed in the PTG, the period of measurement of flow was 10 s. The PTG was connected to a differential pressure transducer. Both transducers were assembled together with connecting valves to ease the calibration and clearing of the esophageal catheter (Pneumomultitest EREMS, Toulouse, France). Total lung resistance was calculated by purposedesigned software using a first order differential equation relating Peso to flow DP = (1/C) × V + R× V% where DP: the change in Peso, C: the static compliance, V and V%: volume and flow, respectively, R: total lung resistance. Respiratory frequency (f) was also retained as an index of the respiratory pattern. Aerosolizations were made through a De Vilbiss nebulizer (Ref 123016 Marquest Medical Products, Englewood CO. USA) connected to a compressor (flow rate= 6 L/min). The aerosols were delivered in a rigid plastic chamber placed over the rat. Aerosol flow rate was 0.1 ml/min. The duration of aerosolization was 10 min. Verapamil was administered at a concentration of 0.01 M (4.9 mg/ml) alone or with NaF at a concentration of either 1 or 0.5 M (42 or 21 mg/ml). The control group received saline at the same speed. Challenges were conducted 30 min after NaF and/or verapamil aerosols with 2.7, 5.5, 11, 21.9, 43.8, 87.7 and 175 mM carbachol solutions aerosolized for 1 min with 3 min intervals between doses. LD50 is the dose of carbachol required to cause the death of five out of 10 rats. In order to estimate the quantity of nebulized fluoride received by each animal, the following parameters were taken into account: the flow rate of the compressor was 6 L/min, the nebulizer produced 0.1 ml/min of aerosol and the rats weighed around 230 g. For 230 g body weight, the estimated tidal volume was about 1 ml, frequency was about 100 breaths per min, and ventilation was 100 ml/min. During the 10 min of aerosolization, the volume of solution delivered to the animal is calculated as: D= V× (A/F) × 10, where D (ml) is the volume of solution delivered to the rat,
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V (L/min) is the ventilation, A (ml/min) is the aerosol production rate, F (L/min) is the flow rate of the air compressor. For instance, over 10 min of nebulization with 1 M NaF and/or 0.01 M verapamil about 71 mmole NaF and/or 0.71 mmole verapamil were inhaled per kilogram. Using nebulization of 99m-Tc pertechnetate (Popa et al., 1984) found that approximately 10% of the radioactivity delivered with the nebulization system was taken up from the trachea and both lungs. Thus, in the present study, about 7 mmol/ kg NaF (0.14 mg F) and/or 0.07 mmol/kg verapamil were assumed to be deposited in the airways for the NaF (1 M)+ verapamil group and half that dose for the NaF (0.5 M) and NaF (0.5 M)+ verapamil groups. The left femoral artery was cannulated for monitoring arterial pressure (BP) and heart rate (HR) (EM-134 pressure transducer, Elema-Schonander, Solna, Sweden). Signals were acquired and analyzed with a computer using Labtech Notebook software (Laboratory Technologies Corporation, Wilmington, MA, USA). At the end of the experiments, i.e. 1 h after the NaF aerosols, rats were exsanguinated and blood samples were collected for measuring the NaF and verapamil concentration in plasma. The plasma concentration of NaF was tested by ionometry and verapamil was tested by gas chromatography coupled with mass spectrometry.
2.2. Protocol 2: NaF applied during the carbachol challenge in rats The procedure was similar to protocol 1 except that rats were first challenged with gradually increasing doses of carbachol up to 11 mM. Then either NaF 0.5 M (n = 8) or NaF 0.25 M (n =10) aerosols were delivered for 1 min followed by the remaining carbachol doses. Control rats received aerosols of normal saline instead of carbachol solutions. The total lung resistance (R) was measured 3 min after each dose of carbachol.
2.3. Protocol 3: effect of NaF on the airway responsi6eness in patients with mild asthma Totally 18 patients were tested for the protective effect of NaF on methacholine provoked
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bronchoconstriction. The human experiment protocol was approved by the ethical committee of the Sousse University Hospital. All participants were informed of the risks and consented to the protocol. Patients were 11 men and 7 women aged 32910 years, suffering from mild asthma. They received less than 2 b2 agonist puffs per week and were free of corticosteroid drugs. No patient had taken any medication for at least 12 h before the study started. Bronchial reactivity was tested with methacholine delivered in aerosol form with an increasing number of puffs. Each patient received a maximum of 15 increasing doses of methacholine from 0 (saline) to 3000 mg. Three min after each dose, a forced maximal expiration was measured with a flowmeter (Minato, Japan) from which the FEV1 was measured. When FEV1 fell by more than 20%, the patient received either an aerosol of 0.5 M NaF, while breathing normally during 15 consecutive cycles, or 0.5 M saline following the same protocol. With a 1 liter tidal volume, the estimated inhaled dose was 1.2× 10 − 4 mole. On the assumption that 10% of the inhaled dose was deposited, the effective received dose was 1.2×10 − 5 mole, i.e. 0.2 mg of fluoride. The nurse performing the forced expiratory maneuver was not aware of the type of aerosol inhaled by the patients. Nine patients were assigned randomly to each group (saline or NaF). Forced maximal expirations were performed 3, 5, 10 and 15 min after the patients received saline or NaF.
2.4. Data analysis In protocols 1 and 2, the results were expressed as mean +6 standard error (SEM). Mean values of total resistance (R) and frequency rate (f), as well as mean values of NaF and verapamil concentrations between control and test groups were compared using Student’s t-test and the Mann– Whitney U-test. As both the student t-test and the ANOVA plus post-hoc test revealed the significant differences (PB 0.05) between the same data pairs, only the results of t-tests are presented. For comparing the mean values between the control
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and experimental groups and among the experimental groups, the variances of the groups were subjected to ANOVA followed by a posthoc analysis (Tukey and Student– Newman – Keuls test) using TADPOLE III software (Elsevier –Biosoft). Statistical significance was set at P B 0.05. In protocol 3, the changes in FEV1 following the methacholine challenge in the groups receiving either NaF or saline were subjected to a two-way ANOVA. The FEV1 values from the same patient at different times were compared using the paired Student’s t-test.
2.5. Chemicals used Carbachol, verapamil, NaF and ketamine were purchased from Sigma (St. Louis, MI, USA), and methacholine from Allerbio (Lavarene, France). NaF was dissolved in distilled water devoid of traces of aluminium.
3. Results
3.1. Protocol 1 Neither verapamil nor NaF aerosol alone had any significant effect on ventilatory or cardiovascular parameters in the rats tested. The LD50 of carbachol of NaF (0.5 M), NaF (1 M) + verapamil and NaF (0.5 M)+ verapamil groups were respectively 1289 42, 1139 26 and 1649 42 mM compared with 179 3 and 229 3 mM of the control and verapamil groups, respectively. The means of maximal doses of carbachol received by NaF (0.5 M), NaF (1 M)+ verapamil and NaF (0.5 M)+ verapamil groups were significantly higher than those of the control and verapamil groups (PB 0.05, Fig. 1). Changes in HR and BP were modest and non significant during aerosolizations in both control and verapamil groups. In the NaF (1 M)+ verapamil group, a 10% decrease in HR (PB 0.05) was observed. After 21.9 mM carbachol aer
Fig. 1. LD50 and maximal survival doses for rats against carbachol challenge. LD50 (open column) and mean values of maximal survival doses (hatched column) of control (CTL), verapamil (VPM), NaF 0.5 M (NaF), NaF (1 M) +verapamil (F1M +VPM) and NaF (0.5 M) + verapamil (F0.5 M + VPM) groups were plotted vs. carbachol concentration. Data are given for 10 Wistar male rats for each group. Vertical bars indicate S.E.M. *PB 0.05 (compared with control group); +P B0.05 (compared with verapamil group).
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Table 1 Mean heart rate (HR) and blood pressure (BP) Before
30 min after verapamil and/or NaF
After 21.9 mM carbachol
Groups
HR/min
BP mmHg
HR/min
BP mmHg
HR/min
BP mm/Hg
Control Verapamil NaF0.5M NaF1 M+verapamil NaF 0.5 M +verapamil
403930 3869 41 4109 23 4019 41 3939 30
107 98 1159 9 1049 10 1079 7 1169 17
413 930 372 914 412 921 360 932a 388 936
105 9 11 108 910 109 99 113 911 115 914
338 921a,b 300 9 29a,b 305 9 32a,b 252 9 16a,b 267 9 30a,b
77 9 9a,b 75 916a,b 679 10a,b 79 98a,b 76 9 12a,b
Effect of verapamil, NaF or NaF+verapamil and carbachol. Mean 9 S.E. a PB0.05 (compared with control). b PB0.05 (compared with 30 min after verapamil and/or NaF).
solization, both HR and BP decreased significantly in all groups (Table 1). Up to 21.9 mM carbachol, R value increased slightly and there were no significant differences between the groups. Above this concentration, R values in the NaF (0.5 M), NaF (1 M)+ verapamil and NaF (0.5 M) + verapamil groups were significantly lower than those in the control and verapamil groups (Fig. 2). Up to the third dose of carbachol, f value did not change significantly compared with control (131.59 5/min). At higher doses of carbachol, f slowed slightly in the NaF and NaF+verapamil groups (75.59 29, 88.09 22 and 78.3914.9/min for NaF 1 M, NaF 0.5 M + verapamil and NaF 0.5 M groups, respectively for 43.8 mM carbachol), but it fell sharply in the control and verapamil groups from 131.59 5 to 479 10.7 and 63.49 19.6/min, respectively at 43.8 mM carbachol (P B0.05 compared with 0.5 M NaF, 0.5 M NaF +verapamil groups). Plasma concentrations of fluoride were 9.29 2.3, and 2.5 91.1 mM in NaF (1 M)+verapamil and NaF (0.5 M)+ verapamil groups respectively vs 0.679 0.14 mM in the control group. The mean plasma concentration of verapamil in the groups receiving this drug was 469 18 nM.
creased in the 0.5 M NaF group with increasing doses of carbachol, whereas it increased in both the control and 0.25 M NaF groups, the differences were significant for the two highest concentrations. In the 0.5 M NaF group, the R values at the last two concentrations of carbachol were not significantly different from the control value recorded before the carbachol challenge.
3.3. Protocol 3 All patients were hyperreactive to methacholine. The least reactive patients received 2000 mg of methacholine while the most reactive patients received 50 mg of methacholine. No bouts of coughing were induced by the inhalation of either
3.2. Protocol 2 Slight but significant increases in R values were observed in all three treatment groups after both the first and second doses of carbachol (Fig. 3). Starting at the fourth dose, NaF aerosol was delivered together with carbachol. R value de-
Fig. 2. Effect of pre-inhaled NaF and/or verapamil on rat airway resistance against carbachol challenge, n =10.
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4. Discussion
Fig. 3. Effect of NaF inhaled during the carbachol challenge on rat airway resistance. Arrow: NaF aerosol. N =10 except for NaF 0.5 M group (n= 8).
0.5 M NaF or 0.5 M NaCl. FEV1 decreased by 25 and 30% in the NaF and saline groups, respectively (P\0.05). After 3 min of either NaF or saline inhalation, no significant alteration in FEV1 was observed. However, a significant increase in FEV1 in the NaF group, but not in the saline group, was observed after 5 min inhalation. The elevated FEV1 in the NaF group persisted for at least 10 min. Although FEV1 also increased significantly in the saline group after 15 min of inhalation, it was still significantly lower compared with that of NaF group (Fig. 4).
Fig. 4. FEV1 alterations during the methacholine challenge and recovery with NaF (filled circle, nine patients), or saline (filled triangle, nine patients). NS: not significant, *PB 0.05, **PB 0.01. P values: paired comparisons between initial fall in FEV and recovery in one given group.
The main findings of this study are: (1) NaF, inhaled with or without verapamil before carbachol challenge, decreased the airway responsiveness resulting in a five-fold increase in the LD50 of carbachol. (2) NaF, inhaled during carbachol challenge, decreased R as compared with the control groups. (3) NaF, inhaled by hyperreactive asthmatic patients during methacholine challenge, had bronchodilating effects consistent with that observed in the rats. Unlike the results of a previous in vitro study (Zhao and Gue´ nard, 1997), no significant difference in resistance was observed between the NaF + verapamil and NaF groups. Moreover, there was no difference in values of airway resistance between the control and NaF (0.5 M) groups. This lack of effect of verapamil associated with NaF was not surprising as the purpose of using this drug was to suppress the bronchoconstrictor effect of NaF observed in the in vitro study. However, this NaF-induced bronchoconstrictor effect was not observed during the in vivo study. Verapamil thus did not seem to be required for an inhibitory action of NaF in vivo. The discrepancy between the in vitro and in vivo studies could be due to differences in species as the in vitro study was conducted in bovine bronchus and the present study was carried out on rats and asthmatic humans. However, other factors such as the experimental protocol, in vivo versus in vitro, and differences in concentration of NaF cannot be ruled out. Fig. 2 shows that in the verapamil group, the dose–response curve of carbachol was, compared with control, shifted to the right in agreement with the results obtained in man with nifedipine (Popa et al., 1984). In our previous in vitro study (Zhao and Gue´ nard, 1997), the active dose of verapamil (10 mM) was similar to that of carbachol, which induced a 2-fold increase in the intra-bronchus pressure developed by smooth muscle contraction. This was reason why we employed this dose of verapamil in the present experiments. The dose inhaled seemed adequate as: (1) HR and BP were not altered by verapamil inhalation and (2) serum concentrations of verapamil were within the usual range in
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blood samples obtained from patients receiving this drug. The 1 M concentration of NaF was used in rats for the following reasons: in the in vitro experiments, the contractile effect of 10 mM carbachol on bovine bronchial smooth muscle was 50% inhibited by 5 mM NaF (Zhao and Gue´ nard, 1997), and so an inhaled concentration of 2000× 10 mM carbachol (20 mM) during 1 min would need 2000×5 mM NaF, i.e. 1 M during 10 min to obtain the same effect. However, as this concentration was hypertonic and led to relatively high serum fluoride concentration, 0.5 M NaF was also tested and produced similar results on carbachol responsiveness experiments. Although the carbachol challenge was standardized, the dose received by the animal depended on ventilation. When the R value increased markedly during the provocation tests, the rats would be hypoventilating when receiving high doses of carbachol, and so the amount of carbachol actually reaching the airway would be smaller than estimated. This could result in a less pronounced increase in R than in the control, verapamil and 0.25 M NaF groups in which R increased sharply. On the other hand, the delivery of carbachol was less affected in the 0.5 M and 1 M NaF groups in which R changed slightly. Since bronchoconstriction would tend to lower the breathing frequency, this variable was also taken into account. The f values decreased more rapidly in both the control and verapamil groups than in the NaF and NaF+ verapamil groups with increasing doses of carbachol, as predicted from the greater increases in R. After administration of 21.9 mM carbachol, significant decreases in both HR and BP in all the groups of rats were observed irrespective of the presence or absence of verapamil, suggesting that carbachol lowered HR either directly or via an interactions between lung and heart, i.e. loaded breathing conditions. As bradycardia was also observed in the groups receiving NaF, which were obviously less loaded by the carbachol-induced bronchoconstriction, a direct effect of carbachol on HR seemed more likely. Although the cellular effects of fluoride were not examined, they warrant discussion as they
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could have an influence in vivo. At physiological or pharmacological (therapeutic supplementation) concentrations, i.e. in the 0.5– 10 mM range (Whitford, 1996) the only reported action is on mineralisation of bones and teeth (Heifetz and Horowitz, 1986). Nothing seems to be known about the effect of fluoride supplementation on respiratory status in contrast to that reported for magnesium salts (Alamoudi, 2000). At toxicological concentrations, i.e. in the mM range, many effects have been described, which depend on three factors, the fluoride concentration, the length of exposure and the associated cation. For example 5 mM NaF has a modest effect on the IL-6 and IL-8 responses of a human epithelial cell line 24 h after addition, which was strongly enhanced by the addition of Al3 + (Refsnes et al., 1999). The AlF-4 complex is an activator of GTPbinding proteins (Leurs et al., 1991). Another study showed that 0.5 mM NaF enhances IL-1 beta mRNA expression from lung lavage cells (Hirano et al., 1999). Such increases in chemokine activities would be expected to increase bronchial reactivity. NaF has also been reported to stimulate adenylyl cyclase activity on smooth muscles (Stadel and Crooke, 1988) and induce NO synthesis (Cushing et al., 1990; Kawase et al., 1996) which would relax bronchi. However, the fact that pyruvate reverses the relaxing effect of NaF in vitro is more in favor of a metabolic relaxing effect of NaF. It should be borne in mind that the above effects were obtained with concentrations and lengths of exposure which are known to be toxic for many organs (Black et al., 1999; Gessner et al., 1994) and are thus of more toxicological than clinical interest. NaF is also an inhibitor of glycolysis that is widely used in biochemistry to prevent glucose consumption in blood samples, or for its bactericidal activity in products such as tooth-paste. It is worthy to note that the inhibitory effect of NaF on the glycolytic enzyme enolase can be observed in the 10 mM range (Curran et al., 1994) on bacteria, which could be of interest in certain lung diseases. In vitro anerobic glycolysis is essential for both the active stress and the sustained contraction as neither KCN nor hypoxia have any influence on carbachol-induced contraction. However, in the presence of NaF, the
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relaxation was found to become sensitive to both factors (Zhao and Gue´ nard, 1997). An inhibitor of glycolysis by reducing ATP production could impair membrane or muscle fiber function, or both. The main ATP consumer of the cell membrane is Na+/K+ATPase, and a reduction of its activity would induce an increase in contractility as does ouabain on cardiac myocytes. Nevertheless, the ATP consumption of Na+/K+ATPase is only 14% of the ATP produced during a vascular contraction (Hellstand et al., 1984), and the major ATP consumer during contraction is thought to be the myosin light chain which maintains contraction (Giembycz and Raeburn, 1992). Whatever the mechanism of the relaxing effect of NaF in vivo, it seems likely that the bronchodilating effect observed was due to a metabolic effect, which warrants further investigation. From the total dose of NaF received by rats, the measured fluoride blood concentrations remained in the pharmacological range, and so should not have toxic effects on organs such as bones, teeth and kidneys, which were supposed to be the most sensitive to fluoride toxicity (Whitford, 1996). In patients, the inhaled dose per kg body weight was relatively less than used in rats, and so blood NaF levels were also likely to be lower. In human adults, the safe maximal oral dose is 2 mg F/day. Our patients received one tenth of this dose from the aerosols. As the aerosol contained concentrated NaF, the concentration of NaF in the epithelium lining was probably high, depending on the amount of aerosol deposited, the water transportation and the diffusion of NaF through the epithelium. In future experiments it should be interesting to estimate NaF concentrations in epithelial and bronchial smooth muscle. Comparing the results obtained from human test with those from animal experiments, some differences in the effects of NaF could be identified i.e. the onset of the action in human asthma patients was slower than that observed in animal experiment and the reversal of the bronchoconstriction was incomplete in human subjects but complete in rats (Fig. 2). The possible reasons for these discrepancies are: (1) spe-
cies and health state differences: in rats respiratory system, the reactions to muscarinic agonist provocation and NaF may differ from human and it is the very reason for us to extend the study from rat to human. In animal experiments, healthy rats were used to test the NaF effect whereas in human study, the NaF effects were tested in asthmatic patients. (2) The dosage of muscarinic agonists and NaF: as a safety concern, metacholine and NaF delivered to patient were much lower than used in rats as calculated per unit of body weight. This approach may affect the potencies and effectiveness of the drugs tested, i.e. the delay of the onset of the effect and the incomplete inhibition of bronchoconstriction. The inhaled NaF did not increase plasma fluoride levels and had no general side effects. However, if NaF is used more frequently its chronic toxicity needs to be considered. The long-term effect of NaF should also be studied together with its preventive effect. It is difficult to assess the value of NaF in the treatment of asthma as this study seems to be the first to demonstrate its bronchodilating effect in vivo. Our findings indicate that it might be worth to develop molecules targeting at the metabolism of bronchial smooth muscle and to manipulate the energy metabolism state. In contrast to vascular smooth muscle (Barron et al., 1998), anerobic glycolysis is essential for the sustained contraction of bronchial smooth muscle, making this metabolic pathway a target for relaxation of bronchi. Unfortunately, no other non-toxic molecule, apart from NaF at low dose, is available at present. In conclusion, the present study provides the first in vivo evidence showing that inhaled NaF reduces the bronchial reactivity to carbachol and relaxes acetylcholine analog-induced bronchoconstriction in both rats and asthmatic patients. As indicated by a previous in vitro study, the bronchodilating effect of NaF was attributed to the inhibition of glycolysis by fluoride. Our findings point to the possibility of relaxing bronchi with a molecule that acts directly on the energy metabolism of bronchial smooth muscle.
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Acknowledgements This study was supported by a grant from ‘Genie Biologique et Medical Aquitaine’. W. Zhao was the recipient of a Visiting Lecturer Scholarship from the ‘Universite´ de Bordeaux II, Faculte´ de Me´ decine Victor Pachon, 33076 Bordeaux, France’. S. Rouatbi was the recipient of a research grant from the Tunisian government. The authors are grateful to P. Techoueyres, A.-M. Lomenech, J.-L. Lachaud and H. Crevel for their technical assistance. We also thank the Service de Pharmacologie et Toxicologie, Centre Regional de Pharmacovigilance, Centre Hospitalier Universitaire de Limoges, France.
References Adeagbo, A.S.O., Triggle, C.R., 1991. Mechanism of vascular smooth muscle contraction by sodium fluoride in the isolated aorta of rat and rabbit. J. Pharm. Exp. Ther. 258, 66– 73. Alamoudi, O.S.B., 2000. Hypomagnesaemia in chronic, stable asthmatics: prevalence, correlation with severity and hospitalization. Eur. Respir. J. 16, 427 –443. Barron, J.T., Barany, M., Gu, L., Parillo, J.E., 1998. Metabolic fate of glucose in vascular smooth muscle during contraction induced by norepinephrine. J. Mol. Cell Cardiol. 30, 709 – 719. Black, M.M., Kleiner, I.S., Bolker, H., 1999. The toxicity of fluoride in man. New York St. J. Med. 49, 1187 – 1188. Curran, T.M., Buckley, D.H., Marquis, R.E., 1994. Quasi irreversible inhibition of enolase of Streptococcus mutans by fluoride. FEMS Microbiol. Lett. 119, 283 –288. Cushing, D.J., Sabouni, M.H., Brown, G.L., Mustafa, S.J., 1990. Fluoride produces endothelium-dependent relaxation and endothelium-independent contraction in coronary artery. J. Pharm. Exp. Ther. 254, 28 –32. Gessner, B.D., Beller, M., Middaugh, J.P., Whitford, G.M., 1994. Acute fluoride poisoning from a public water system. New Engl. J. Med. 330, 95 –99.
253
Giembycz, M.A., Raeburn, D., 1992. Current concepts on mechanisms of force generation and maintenance in airway smooth muscle. Pulmonary Pharmacol. 5, 279 – 297. Heifetz, S.B., Horowitz, H.S., 1986. Amounts of fluoride in self-administered dental products: safety consideration for children. Pediatrics 77, 876 – 882. Hellstand, P., Jorup, C., Lydrup, M.L., 1984. Oxygen consumption, aerobic glycolysis and tissue phosphagen content during activation of the Na+/K+ATPase pump in rat portal vein. Pflu¨ gers Arch. 401, 119 – 124. Hirano, S., Ando, M., Kanno, S., 1999. Inflammatory responses of rat alveolar macrophages following exposure to fluoride. Arch. Toxicol. 73, 310 – 315. Kawase, T., Oguro, A., Orikasa, M., Burns, D.M., 1996. Characteristics of NaF-induced differentiation of HL-60 cells. J. Bone Miner. Res. 11, 1676 – 1687. Leurs, R., Bast, A., Timmerman, H., 1991. Fluoride is a contractile agent of guinea pig airway smooth muscle. Gen. Pharmacol. 22, 631 – 636. Mayes, P.A., 1987. Glycolysis and oxidation of pyruvate. In: Granner, D.K., Mayes, P.A., Murray, P.K., Rodwell, W.W. (Eds.), Harper’s Biochemistry, 21st ed. Section 2: Bioenergetics and metabolism of sugar and lipids. Lange Medical, San Mateo, pp. 177 – 184. Popa, V.T., Somani, P., Simon, V., 1984. The effect of inhaled verapamil on resting bronchial tone and airway contractions induced by histamine and acetylcholine in normal and asthmatic subjects. Am. Rev. Respir. Dis. 130, 1006 – 1013. Ratz, R.H., Blackmore, P.F., 1990. Differential activation of rabbit femoral arteries by aluminium fluoride and sodium fluoride. J. Pharmacol. Exp. Ther. 254, 514 – 520. Refsnes, M., Becher, R., Lag, M., Skuland, T., Scwarze, P.E., 1999. Fluoride-induced interleukine-6 and interleukine-8 synthesis in human epithelial cells. Hum. Exp. Toxicol. 18, 645 – 652. Stadel, J.M., Crooke, S.T., 1988. Differential effects of fluoride on adenylate cyclase activity and guanine nucleotide regulation of agonist high affinity receptor binding. Biochem. J. 254, 15 – 20. Whitford, G.M., 1996. The metabolism and toxicity of fluoride, second, reversed ed. Monographs in Oral Science, 16, pp. 1 – 154. Zhao, W., Gue´ nard, H., 1997. The inhibitory effect of fluoride on carbachol-induced bovine bronchial contraction. Respir. Physiol. 108, 171 – 179.
