Airway Responses to Inhaled Ouabain in Subjects With and Without Asthma

Airway Responses to Inhaled Ouabain in Subjects With and Without Asthma

KRISHNA P. AGRAWAL, M.D., Research Fellow in Physiology*; CHARLES E. REED, M.D., Division of Allergic Diseases and Internal Medicine; ROBERT E. HYATT, M.D., Division of Thoracic Diseases and Internal Medicine; WAYNE E. IMBER, M.D., Resident in Allergyt WILLANE S. KRELL, M.D., Resident in Thoracic Diseases%

Challenges with ouabain and histamine were performed a week apart in 10 patients with asthma and 5 normal subjects. Concentrations were increased cumulatively until specific airway conductance decreased by 30% or the maximal concentration of 1.0% was reached. At low concentrations, ouabain induced bronchodilatation in six patients who had asthma. Bronchodilatation gradually decreased with increasing concentrations and was followed by bronchoconstriction in two patients with asthma who had high airway sensitivity to histamine. Ouabain caused only bronchoconstriction in three patients with severe asthma. The normal subjects showed mild bronchodilatation or no response to ouabain. Several possible biochemical mechanisms may be responsible for the bronchodilatory response to low doses of ouabain, such as stimulation of adenylate cyclase or (Na\K + )-adenosine triphosphatase. The absence of a bronchodilatory response to ouabain in patients with severe asthma suggests an impairment in the activity of these enzymes.

Recently, Nath and associates 1 reported a positive correlation between in vivo airway responsiveness to histamine and tracheal (Na+,K+)adenosine trisphosphatase (ATPase) activity in guinea pigs. They suggested that (Na+,K+)-ATPase *Mayo Graduate School of Medicine, Rochester, Minnesota. Present address: Vallabhbhai Patel Chest Institute, University of Delhi, Delhi, India. tMayo Graduate School of Medicine, Rochester, Minnesota. Present address: Rancho Encino Hospital, Encino, California. JMayo Graduate School of Medicine, Rochester, Minnesota. Present address: Harper Hospital, Detroit, Michigan. This investigation was supported in part by Research Grant HL-21584 from the National Institutes of Health, Public Health Service, and by a grant from the Mayo Foundation. Dr. Agrawal was supported by a Parker B. Francis Foundation Fellowship. Address reprint requests to Dr. R. E. Hyatt, Division of Thoracic Diseases, Mayo Clinic, Rochester, MN 55905. Mayo Clin Proc 61:778-784, 1986

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functions as a homeostatic mechanism by preventing Na + loading of the cell. Subsequently, we observed a positive correlation between airway responsiveness to histamine and ouabain, an inhibitor of (Na+,K+)-ATPase, in intact conscious guinea pigs. 2 In the current study, we examined the relationship between airway responsiveness to ouabain and histamine in subjects with and those without asthma. On the basis of previously published findings, 1,2 ouabain was assumed to provide an indirect estimate of (Na+,K+)-ATPase activity and its possible alteration in patients with asthma.

SUBJECTS A N D METHODS Histamine and ouabain challenges were conducted 1 week apart in 5 subjects without asthma (Table 1) and in 10 patients with asthma (Table 2).

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Table 1.—Baseline Pulmonary Function and Airway Responsiveness to Histamine in Five Subjects Without Asthma* Baseline pulmonary function SGaw Inhalation Age (yr) Visit Hist ED 30 Vmaxso (1/cm H 2 0-s) challenge (L/s) and sex no.f (mg/ml) Case Ouabain 29 M 1 1 0.15 (89)J 8.18(131)t Histamine 7.80 2 0.15 >10 Ouabain 4.59 (78) 28 M 1 0.16 (95) 2 Histamine 4.45 2 0.15 >10 Ouabain 6.85 (123) 29 M 1 3 0.19(112) Histamine 6.99 2 0.19 >10 Ouabain 5.57 (143) 37 F 1 0.26 (154) 4 Histamine 5.70 2 0.23 >i'o Ouabain 4.60 (98) 34 M 1 5 0.17(101) Histamine 4.21 2 0.16 >10 *Hist ED30 = concentration of histamine that produced a 30% decrease in SGaw; SGaw = specific airway conductance; Vmaxso = maximal expiratory flow at 50% of the vital capacity. tVisits were scheduled 1 week apart. JValues in parentheses are percent of predicted normal.

Normal values for specific airway conductance (SGaw) and for flow were obtained from publications by Krell and colleagues 3 and by Knudson and co-workers,4 respectively. On the basis of the clinical history and pulmonary function tests, 7 of our 10 patients were considered to have mild asthma and 3 to have severe asthma. These three patients had required corticosteroid therapy, but two were not currently on such a regimen. Theophylline was withheld overnight in two patients (cases 8 and 9). One patient (case 10) had taken his daily morning dose of prednisolone (5 mg). No other subjects were taking any medication. Pulmonary function tests were performed before inhalation challenge with either drug. Inhalation challenges were done with the subjects in a constant-volume body plethysmograph. We used an open-loop method for the measurement and continuous monitoring of changes in SGaw, as described recently. 3 Three to five breaths were recorded on tape. At a later time, the breaths were measured by two technicians who had no information about what substance was being administered. The mean of the two measurements was used for analysis. Aerosols were generated by passing an air current at 20 psi through a nebulizer (DeVilbiss 645) that contained a solution of histamine (histamine diphosphate; Sigma Chemical Company, St. Louis, Missouri) or ouabain (ouabain octahydrate; Sigma) in phosphate-buffered saline and were delivered through a newly designed valve. The subjects could not tell which solution was being administered. Concentrations of both drugs used

for inhalation challenges ranged from 0.004 to 1.0% (0.04 to 10 mg/ml). Five puffs of 0.6-second duration of each concentration were routinely delivered during inspiration by using a manually triggered dosimeter. In three subjects (only one of whom had asthma), up to 20 puffs of 1.0% ouabain solution were delivered. (Ouabain solutions of higher strength could not be prepared because of poor solubility.) Four patients with asthma were given phosphate-buffered saline repeatedly by aerosol to test for any nonspecific cumulative effect. No change in SGaw was found. Log concentration-response curves were plotted to determine the concentration of histamine that produced a 30% decrease in SGaw (Hist ED 30 ). The effects of the final dose of ouabain on SGaw lasted from 1 to 3 hours. The changes in SGaw in response to ouabain in subjects without asthma and in various groups of patients with asthma and the prechallenge pulmonary function tests and airway responsiveness to histamine were compared by using analysis of variance at each concentration of ouabain. Homogeneity of variances was determined by using Bartlett's test. At concentrations for which a significant difference existed between the groups, the least significant difference t test for any two groups was performed. This test is identical in form to the two-sample t test except that it uses the pooled variance estimate obtained over all groups, not just the two being compared. The degrees of freedom were those associated with the pooled variance across all groups. The significance level was set at P<0.05.

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AIRWAY RESPONSES TO INHALED OUABAIN

RESULTS Baseline Pulmonary Function Tests. —Tables 1 and 2 present the prechallenge (baseline) values of maximal expiratory flow at 50% of the vital capacity (Vmax5o) and SGaw in 5 subjects without asthma and 10 with asthma. The subjects without asthma had normal Vmax5o and SGaw values; the patients with mild asthma had some airway obstruction, and those with severe asthma had substantially abnormal results of both tests. Thus, the results of the pulmonary function tests were in general agreement with the clinical evaluation. Airway Responses to Histamine.—Airway responsiveness to histamine was arbitrarily classified as low, moderate, or high. Responsiveness was low in all subjects without asthma (Hist ED30 more than 10 mg/ml), moderate in five patients with mild asthma (Hist ED30 = 0.48 to 1.5 mg/ml), and high in two with mild and all three with severe asthma (Hist ED30 less than 0.25 mg/ml). These results indicate that airway reactivity need not be directly correlated with the severity of asthma

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as judged by clinical and pulmonary function criteria. Airway Responses to Ouabain.—Figure 1 shows the effect of inhalation of ouabain on SGaw in five subjects without asthma. These subjects had low airway reactivity, as reflected by their Hist ED30 values (Table 1). Ouabain produced mild but significant bronchodilatation at the 0.031 and 0.25% concentrations. In two normal subjects, pronounced sensory effects were observed after 20 puffs of 1.0% ouabain; they had severe coughing and complained of throat irritation and rawness in the anterior part of the chest. We noted no consistent effects on heart rate or blood pressure. Figure 2 shows the effect of inhalation of ouabain on five patients with mild asthma who had moderate airway reactivity (Hist ED30 = 0.48 to 1.5 mg/ml). In four of these patients, the response was definite bronchodilatation, as indicated by an increase in SGaw of up to 50%. One patient (case 1 in Table 2) showed no response. For this group, the bronchodilatory response was significant at con-

Table 2.· -Baseline Pulmonary Function and Airway Responsiveness to Histamine in 10 Patients With Asthma* Baseline pulmonary function Age (yr) Visit Inhalation Clinical Vmaxso SGaw at FRC Hist ED30 (1/cm H 2 Os) (mg/ml) (L/s) Case and sex challenge no.f status 1.40 20 M Histamine 3.26 (56)t 1 1 0.12 (71)t Mild asthma 1.90 0.06 Ouabain 2 2.05 (46) 2 0.12 (71) 55 M Mild asthma 1 Ouabain 1.25 0.12 Histamine 2.25 2 1.50 3 0.14 (83) 27 M Mild asthma 1 Histamine 3.28 (52) 3.10 0.13 Ouabain 2 0.58 4 0.10(59) 45 F Mild asthma 1 Histamine 1.65(43) 0.09 1.40 Ouabain 2 0.48 5 0.10(59) 31 M Mild asthma 1 Histamine 3.20 (55) 0.10 3.43 Ouabain 2 0.16 6 0.08 (47) 60 M Mild asthma 1 Histamine 3.80 (87) 0.08 3.60 Ouabain 2 7 0.05 (30) 2.26 (44) 28 M Mild asthma 1 Ouabain 0.10 0.06 Histamine 2.35 2 0.21 8 0.03 (18) 36 F Severe asthma; 1 Histamine 1.34 (33) 0.03 1.10 corticosteroids Ouabain 2 needed 9 0.05 (30) 0.55 (15) 46 F Severe asthma; 1 Ouabain 0.03 0.08 corticosteroids 2 Histamine 0.46 needed 0.04 (24) 0.45 (9) 10 Ouabain Severe asthma; 1 42 M 0.07 0.02 2 Histamine 0.64 corticosteroiddependent *FRC = functional residual capacity. For explanation of other abbreviations, see first footnote to Table 1. tVisits were scheduled 1 week apart. JValues in parentheses are percent of predicted normal.

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Fig. 1. Effect of inhalation of ouabain administered as an aerosol to five subjects without asthma who had low airway reactivity (see text). Up to concentrations of 1.0%, five puffs of ouabain were inhaled; for delivery of higher doses, the number of puffs was doubled. Ordinate shows change in specific airway conductance (SGaw) relative to its value determined after challenge with phosphate-buffered saline. W/ V = weight/ volume.

Fig. 3. Effect of inhalation of ouabain on two patients with mild asthma and high airway sensitivity to histamine (see text). Bronchodilatation occurred in response to low doses of ouabain, but bronchoconstriction occurred with five puffs of 1.0% ouabain. The one patient (open circles) who inhaled 10 more puffs of 1.0% ouabain had a severe asthmalike attack. SGaw = specific airway conductance; W/V =■ weight/volume.

centrations of 0.031 to 1.0%. Bronchoconstriction was observed in one patient (case 5 in Table 2) after 20 inhalations of 1.0% ouabain. He coughed, wheezed, and had some expectoration. The effect of inhalation of ouabain on two patients with mild asthma who had high airway reactivity (Hist ED30 less than 0.25 mg/ml) is shown in Figure 3. These patients experienced significant bronchodilatation only at the 0.031%

dose of ouabain. Bronchoconstriction tended to develop after five puffs of 1.0% ouabain had been taken. One patient who took 10 more inhalations had a severe attack of asthma. He complained of severe throat irritation and a productive cough; these symptoms persisted for 8 to 10 hours. Inhalation of ouabain in three patients with severe asthma who had high airway reactivity (Hist ED30 less than 0.25 mg/ml) caused some bronchoconstriction at doses higher than 0.062% (Fig. 4), although the group change was not sig­ nificantly different from baseline. The bronchoconstrictive effect was most notable in the corticosteroid-dependent patient, who experi­ enced an attack of asthma that lasted for 10 to 12 hours. In another patient, bronchoconstriction increased during bouts of coughing. This finding contrasts sharply with the results obtained in two subjects without asthma who had no bronchocon­ striction during severe coughing. The means and standard deviations of changes in SGaw in the various groups at differing concen­ trations of ouabain are shown in Figure 5. The 1.0% data are for the first five puffs of ouabain. In one patient who was not challenged with 1.0% ouabain, the change in SGaw at this concentra­ tion was plotted as that at 0.5% because the response seemed to have reached a plateau (Fig. 4). In comparison with subjects without asthma, those who had mild asthma and moderate airway

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Fig. 2. Effect of inhalation of ouabain on patients with mild asthma and moderate airway sensitivity to histamine (see text). In four of these five patients, notable bronchodilatation occurred after inhalation of up to five puffs of 1.0% ouabain. In one subject (open circles) who was given 20 puffs of 1.0% ouabain, specific airway conductance (SGaw) decreased sub­ stantially. W/ V = weight/ volume.

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Fig. 4. Effect of inhalation of ouabain on three patients with severe asthma who needed corticosteroids. These patients had high airway sensitivity to histamine (see text). Ouabain caused bronchoconstriction at all dose levels higher than five puffs of 0.062%. Bronchoconstrictive effect was pronounced in the corticosteroid-dependent patient (closed circles). Coughing increased bronchoconstriction in another patient (open rectan­ gles). SGaw - specific airway conductance; W/V = weight/ volume.

reactivity showed significant relaxation at dose levels from 0.031 to 1.0%. In comparison with normal subjects, those with mild asthma and high airway reactivity had significant bronchoconstriction at high (1.0%) concentrations of ouabain. In patients who had severe asthma and high

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Fig. 5. Effect of inhalation of ouabain in various groups: normal subjects (open circles), patients with mild asthma and moderate airway reactivity (open rectangles), patients with mild asthma and high airway reactivity (closed circles), and patients with severe asthma and high airway reactivity (closed rectangles). Specific airway conductance (SGaw) values are plotted as means and standard deviations (vertical lines). * - significant differences (P<0.05) between the severe and mild asthma groups.

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airway reactivity, bronchoconstriction was significantly worse than in those without asthma at dose levels of ouabain from 0.250 to 1.0%. In Figure 5, we have indicated the doses at which the differences between the patients with severe asthma and those with mild asthma were significant. Thus, the severely affected group was significantly different from the group with mild asthma and moderate airway reactivity at all dose levels and from the patients with mild asthma and high reactivity at doses of 0.124 to 0.50%. The only difference between the two groups with mild asthma was noted at the 1.0% dose of ouabain.

DISCUSSION The results of this study are consistent with the hypothesis that (Na+,K+)-ATPase is abnormal in patients who have asthma. They do not, however, provide a simple explanation of the type of cell involved or of the enzyme abnormality. Depolarization of cell membranes increases Ca++ flow into the cell5 and thereby increases intracellular Ca++. An increase in intracellular Ca++ would also occur as a result of Na + loading of the cell, which would decrease Ca++ efflux by Na+Ca++ exchange. An increase in intracellular Ca++ could result in contraction of smooth muscles, production of mucus, or release of a mediator, depending on the type of cell involved. Subjects without asthma show an inconsistent bronchomotor response to inhalation of ouabain; more data on normal subjects would be of interest. In patients with asthma, the airway response to ouabain is related to the severity of the asthma. Bronchodilatation is the predominant response to ouabain in patients who have mild asthma and moderate airway reactivity (Fig. 2); we have no explanation for the absence of a response in one of our patients in this category. Bronchoconstriction occurs after administration of moderate doses of ouabain in patients who have severe asthma and high airway reactivity (Fig. 4). A biphasic response occurs in patients who have mild asthma and high histamine responsiveness (Fig. 3). Apart from these motor effects, ouabain given in high doses produces substernal burning and coughing, results that suggest that it acts on sensory neurons. A well-defined action of ouabain is inhibition of (Na+,K+)-ATPase,6 an action that would lead to

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membrane depolarization. 7 The depolarization of nerve cell membranes, which occurs predominantly in unmyelinated fibers, 89 may stimulate C-fiber terminals including type J receptors. The stimulation of these receptors could produce coughing and unpleasant respiratory sensations. 10 The question arises whether the bronchoconstrictive effects of ouabain might be due to stimulation of vagal reflexes. The bronchodilatation that occurred to some degree in all our patients except those with the most severe asthma is evidence that contradicts this explanation, but a definitive answer necessitates performance of additional experiments to determine whether atropine would alleviate the bronchoconstriction. Ouabain has been shown to cause depolarization and contraction of smooth muscle in vitro 5,11 and a decrease in SGaw in intact conscious guinea pigs. 2 The airway responses to ouabain and histamine in guinea pigs in vivo were found to be positively correlated. If the same effect occurred in humans, then the higher the airway responsiveness to histamine, the lower should be the dose of ouabain necessary to elicit a bronchoconstrictive response. In our comparisons of subjects with and those without asthma, such a relationship was found. Although a decrease in SGaw was not noted in normal subjects who were the least responsive to histamine (Fig. 1), 20 puffs of 1.0% ouabain produced about a 30% decline in SGaw in one patient with mild asthma and moderate airway reactivity (Fig. 2). Five puffs of 1.0% ouabain caused about a 20% decrease in SGaw in two patients with mild asthma and high airway reactivity, and an additional 10 puffs produced a 40% decline in SGaw in one of these patients (Fig. 3). Bronchoconstriction began at a ouabain dose of 0.25% in patients who had severe asthma and high airway reactivity (Fig. 4). The bronchodilatory effect of ouabain detected in patients with mild asthma was surprising. Such a response was not seen in guinea pigs with hyperreactive airways, and indeed it is difficult to explain. This effect was striking in patients who had mild asthma and moderate airway reactivity, especially when compared with the response in those who had severe asthma (Fig. 5). If the primary effect of ouabain is on smooth muscle, we can speculate about the following possible mechanisms.

AIRWAY RESPONSES TO INHALED OUABAIN

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Redistribution of Intracellular Ca+*.—Re12 cently, Bose and associates reported that ouabain caused relaxation of guinea pig teniae coli that had been stimulated to contract with carbachol. They showed that relaxation was accompanied by redistribution of CA++, which was deposited on the inner surface of the plasma membrane. Ouabain Stimulation ofAdenylate Cyclase With Increase in Tissue Levels of Cyclic Adenosine Monophosphate.—The levels of cyclic adenosine monophosphate in various tissues increase in response to administration of ouabain. 1 3 1 9 Stimulation of adenylate cyclase by ouabain could be an underlying mechanism. 1 3 1 5 Stimulation of (Na+,K+)-ATPase at Low Concentrations of Ouabain.—Ouabain has been reported to stimulate (Na+,K+)-ATPase at low concentrations. 20,21 The dose level at which (Na+,K+)-ATPase inhibition by ouabain begins to occur depends on the microenvironment of the enzyme. For example, it is affected by the K+ concentration in the extracellular fluid.21 Disinhibition by Ouabain of the Inhibitory Effect of Lysophosphatidyl Choline on (Na\K*)-ATPase.—The inhibitory effect of lysophosphatidyl choline on (Na + ,K + )-ATPase has been demonstrated in various tissues. 22-26 Lysophosphatidyl choline-induced inhibition of (Na+,K+)-ATPase has also been shown to be reversed by low doses (25 to 50 nM) of ouabain. 24 In view of the observation that serum levels of lysophosphatidyl choline are significantly higher than normal in patients with asthma, 27,28 it is not surprising that (Na+,K+)-ATPase activity has been found to be low in the white blood cells of these patients. 29 Therefore, disinhibition by ouabain could lead to increased activity of (Na+,K+)ATPase and bronchodilatation. Thus, ouabain could produce relaxation of smooth muscles by a variety of mechanisms. If, however, (Na+,K+)-ATPase activity is low in the airway smooth muscles of patients with asthma, as suggested by Mehta and colleagues, 29 stimulation of (Na+,K+)-ATPase may be the major mechanism underlying the consistent bronchodilatory effect of ouabain in patients with mild asthma. The failure of low doses of ouabain to produce bronchodilatation in patients who have severe asthma is interesting. This finding may be due to alterations in the phospholipid microenvironment of adenylate cyclase or (Na+,K+)-ATPase (or both).

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AIRWAY RESPONSES TO INHALED OUABAIN

Patients with asthma who fail to respond with bronchodilatation to ouabain inhalation challenge may have gross alterations in tissue enzyme activity associated with their severe asthma. In summary, these studies suggest that (Na+,K+)-ATPase activity is altered in patients who have asthma. Additional studies are necessary to determine whether this altered enzyme activity is basic to the pathogenesis of asthma. ACKNOWLEDGMENT We thank Daniel P. Olson for fabricating the aerosol delivery valve, Catherine M. Swee and Darreil L. Loeffler for technical assistance, Gene M. Peters for technical supervision, Patrick W. Welsh for coordination of patients, Patricia A. Muldrow for secretarial help, Susan J. Gunst, Ph.D., for reviewing the manuscript, and Kenneth P. Offord for statistical advice.

REFERENCES

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