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Background We have previously reported that inhaled heparin prevents allergic bronchoconstriction in sheep and inhibits the anti-IgE-mediated release of histamine from mast cells in vitro. Since the release of such mediators has been implicated in exercise-induced asthma, we investigated whether inhaled heparin could also attenuate the bronchoconstrictor response in this disease.
Methods On five days we studied 12 subjects with a history of exercise-induced asthma. On day 1 they underwent a standardized exercise challenge on a treadmill to document the presence of exercise-induced asthma. Minute ventilation was estimated with a calibrated respiratory inductive plethysmograph. The workload was increased until the heart rate reached 85 percent of the predicted maximal value, and was sustained for 10 minutes. The magnitude of bronchoconstriction was assessed by measuring specific airway conductance before and after the exercise. On day 2 the partial-thromboplastin time was measured in plasma obtained before and after the subjects inhaled a nebulized solution of heparin (1000 U per kilogram of body weight). On days 3 through 5 the subjects were pretreated with 4 ml of inhaled heparin (1000 U per kilogram), cromolyn sodium (20 mg), or placebo according to a single-blind, randomized, crossover design and underwent exercise challenge 45 minutes later. To exclude the possibility that heparin had any direct effect on airway smooth muscle, bronchial provocation with histamine was induced in five subjects on two further days after pretreatment with either heparin or placebo.
Results Inhaled heparin and cromolyn sodium had no effect on specific airway conductance at base line, but did attenuate the exercise-induced decreases in this variable: the mean (±SE) maximal decrease five minutes after exercise was 9 ±5 percent after pretreatment with heparin, as compared with 22 ±5 percent after pretreatment with cromolyn and 35 ±2 percent after pretreatment with placebo. Heparin did not change the partial-thromboplastin time and did not modify the bronchoconstrictor response to histamine.
Conclusions Inhaled heparin prevents exercise-induced asthma without influencing histamine-induced bronchoconstriction. This non-anticoagulant action of heparin is more likely to be related to a modulation of mediator release than to a direct effect on smooth muscle.
Stimulus-secretion coupling is important in mast-cell-mediated reactions; both immunologic and nonimmunologic stimuli can degranulate mast cells, and multiple signaling pathways control this process10,11. Stimulation of mast cells starts a series of cellular events (including the breakdown of inositol phospholipids) and culminates in mediator release. The breakdown of inositol phospholipids leads to the generation of 1,4,5-inositol triphosphate, which by binding to receptors on the endoplasmic reticulum can cause internal release of calcium in mast cells and many other cells10,11,12,13. Inositol triphosphate can also degranulate mast cells and cause histamine release, thus underscoring the importance of this molecule in mediator release11.
Heparin acts as a specific blocker of inositol triphosphate receptors and inhibits inositol triphosphate-mediated calcium release in various types of cells13,14,15,16. We have proposed that heparin may block inositol triphosphate receptors in mast cells and, by interfering with signal transduction, may modulate mast-cell degranulation and mediator release17. Recent studies support this hypothesis and have demonstrated that heparin can attenuate antigen-induced bronchoconstriction in sheep and human subjects with asthma and can inhibit histamine release from isolated human mast cells17,18,19,20,21. Heparin has also been shown to prevent histamine release from blood cells, which is induced by antigen, trypsin, and other proteases22. Since mediator release may be involved in exercise-induced asthma, it is possible that heparin may be protective in this condition. In the present investigation we studied the effect of inhaled heparin on this disorder and compared its efficacy with an agent known to stabilize mast cells, cromolyn sodium.
Methods
Subjects
Twelve asymptomatic subjects (eight male and four female subjects) with a history of exercise-induced asthma and a documented bronchoconstrictor response to exercise participated in the study. All the subjects (age range, 15 to 41 years; mean, 27) were nonsmokers, without a history of heart disease or a recent history of upper respiratory tract infection. They were not taking any oral medicines or inhaled corticosteroids, and beta-agonist inhalers were withheld for at least 24 hours before each study day. Written informed consent was obtained from each subject before the study, and the research protocol was approved by the institutional review board for experiments in humans.
Pulmonary Measurements
Pulmonary function was assessed with a spirometer, and airway resistance and thoracic gas volume were measured with a calibrated body plethysmograph (Collins, Braintree, Mass.) according to the method of DuBois et al23,24. Each data point was the average of at least three to five reproducible measurements (variability, <5 percent). Specific airway conductance (SGaw) was calculated by dividing the reciprocal of airway resistance by the thoracic gas volume at which airway resistance was measured. The degree of asthma was defined on the basis of the measurements of SGaw.
Minute ventilation was measured with a respiratory inductive plethysmograph (Respigraph, Non-Invasive Monitoring Systems, Miami Beach, Fla.), because breathing through a mouthpiece with the nose clipped increases ventilation substantially above the level measured during natural breathing (no mouthpiece or nose clip)25,26. Respibands (elastic transducer bands of a respiratory inductive plethysmograph, which measures changes in the cross-sectional area of the rib cage and abdomen) were applied on the thorax just above the nipple line and on the abdomen at the level of the umbilicus. The plethysmograph was calibrated according to the Qualitative Diagnostic Calibration procedure27 while each subject stood on the treadmill. Data were collected for five minutes to obtain base-line measurements of the average respiratory rate, tidal volume, and minute ventilation. The subject then jogged or ran on the treadmill, and data were collected again during the last five minutes of exercise. Toward the end of the exercise period, the subject performed tidal breathing with the spirometer for five to seven breaths while maintaining the level of exercise. The accuracy of the measurement of tidal volume with the plethysmograph was compared with that of measurement of this variable with the spirometer; a calibration error of less than 10 percent at rest and less than 20 percent during exercise was considered acceptable.
Exercise Testing
Exercise testing was performed on a treadmill, and the heart rate was monitored with an electrocardiograph. Exercise challenge was carried out by gradually increasing the workload through increasing the speed or the angle of inclination (or both) until 85 percent of the predicted maximal heart rate was achieved. The subjects were then asked to continue exercising at that workload for 10 minutes. Minute ventilation was measured before exercise and during the last five minutes of exercise. The bronchoconstrictor response to exercise was assessed by measurement of SGaw before and immediately after exercise and then every 5 minutes for 30 minutes or until the SGaw returned to the level before exercise.
Aerosol Delivery and Measurement of Partial-Thromboplastin Time
A solution of heparin, cromolyn sodium, or placebo was administered as a constant-flow aerosol during tidal breathing; each solution was aerosolized with a disposable raindrop medication nebulizer (Puritan Bennett, Lenexa, Kans.) during a 20-minute period. The mass median aerodynamic diameter of droplets discharged from the nebulizer was estimated to be 4.5 microm (geometric SD, 2.1), as measured by a seven-stage Anderson cascade impactor. During tidal breathing, the subjects inhaled the aerosol from the nebulizer through a short mouthpiece during inspiration. On the basis of the distribution of the droplets according to size and the pattern of tidal breathing, as well as the volume of solution actually nebulized (calculated by subtracting the weight of the nebulizer before nebulizations from the weight after nebulization), the dose deposited in the lungs was expected to be 10 to 11 percent of the dose placed in the nebulizer, as previously demonstrated28.
The partial-thromboplastin time was estimated in plasma isolated from a 5-ml sample of venous blood analyzed in the hematology laboratory according to standard methods.
Histamine Delivery
Solutions of histamine (Sigma, St. Louis) were prepared fresh daily. The histamine was diluted in phosphate-buffered isotonic saline solution and delivered by nebulizer (DeVilbiss No. 644, Somerset, Pa.); the mass median aerodynamic diameter of the droplets was 3.9 microm (geometric SD, 2.4). To induce bronchial provocation, the nebulizer was attached to a dosimeter, which consisted of a breath-activated solenoid valve and a source of compressed air (pressure, 20 psi [1.4 atm]). When triggered by the subject's inspiratory effort, the solenoid valve was set to remain open for 0.6 second during inhalation to allow the compressed air to flow through the nebulizer, dispersing an average of 0.023 ml of the solution with each breath. The aerosolized material was delivered from the end-expiratory position through the course of a submaximal inspiratory effort. After the base-line SGaw was measured, the subjects inhaled five breaths of the saline diluent, and the measurements were repeated after a two-minute interval. A dose-response curve for histamine was then established: the subjects took five inhalations of each of the dilutions of histamine solution at intervals of five minutes; the concentration of the first dilution was 0.075 mg per milliliter, and the concentrations of the subsequent dilutions were increased in a doubling manner. Bronchial provocation was stopped when the SGaw fell by at least 50 percent from the value recorded after inhalation of the diluent, or when the maximal concentration of 5 mg per milliliter was reached. The SGaw was then plotted against the cumulative dose of histamine, expressed in breath units; one breath unit was defined as one inhalation of a preparation containing 1 mg of histamine per milliliter. The results were expressed as the cumulative provocation dose of the histamine that decreased the SGaw by 50 percent (PD). At the end of each experiment, the subjects took two inhalations of albuterol to reverse any residual bronchoconstriction.
Preparation of Agents
Heparin sodium (injection USP, in bacteriostatic injection water) derived from porcine intestinal mucosa (Elkins-Sinn, Cherry Hill, N.J.) was used undiluted as an aerosol with a concentration of 20,000 USP units per milliliter. Cromolyn sodium solution (20 mg) was reconstituted in 4 ml of bacteriostatic injection water (Intal, Fisons Pharmaceutical, Loughborough, United Kingdom). The bacteriostatic injection water, which was the vehicle for the heparin, cromolyn, and placebo, contained 9.5 mg of benzyl alcohol per milliliter. The nebulized volume of all three solutions was 4 ml.
Experimental Protocol
Exercise Study
All 12 subjects were studied on five days at least three days apart. Exercise challenge was carried out in an air-conditioned room with constant temperature and humidity. On day 1, after base-line pulmonary-function tests were completed, a control challenge was performed as described above (Exercise Testing) to document the degree of exercise-induced asthma. In all 12 subjects the SGaw decreased more than 30 percent. For each subject the workload determined on the initial screening day was used in the subsequent experiments. On day 2, venous blood (5 ml) was obtained for the measurement of the partial-thromboplastin time in plasma, which was determined before the administration of heparin (1000 U per kilogram of body weight; maximal total dose, 80,000 U) and one hour and three hours after administration. This dose of heparin had completely inhibited antigen-induced bronchoconstriction in sheep17. On days 3, 4, and 5, each subject underwent a standardized exercise challenge for 10 minutes, 45 minutes after inhaling aerosolized heparin (1000 U per kilogram), cromolyn sodium (20 mg), or a placebo solution (vehicle) in a single-blind, randomized, crossover design. SGaw was measured before and 45 minutes after the administration of placebo, cromolyn, or heparin and serially for 30 minutes after the exercise challenge.
Histamine Challenge
On two additional days, five subjects underwent histamine challenge after they inhaled heparin (1000 U per kilogram) or placebo according to a single-blind, randomized, crossover design. The PD values for histamine on days of heparin administration were compared with those on days of placebo administration.
Statistical Analysis
Data are expressed as means ±SE. Values for SGaw were analyzed by a two-way analysis of variance with repeated measures, followed by the Newman-Keuls pairwise comparison for identifying significant differences between pairs. A paired t-test was used to compare the effect of heparin on histamine PD values with that of placebo. A P value of less than 0.05 was considered to indicate statistical significance29.
Results
Base-Line Pulmonary Function
Pulmonary function was normal in all 12 subjects at base line (Table 1). The values for SGaw were comparable on three experiment days, and neither cromolyn nor heparin had a significant effect (SGaw before and after placebo administration, 0.17 ±0.02 and 0.17 ±0.02 liter per second per centimeter of water per liter; before and after heparin, 0.16 ±0.02 and 0.17 ±0.04 liter; and before and after cromolyn, 0.16 ±0.03 and 0.16 ±0.03 liter).
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Aerosolized heparin did not cause an increase in the plasma partial-thromboplastin time in the 10 subjects in whom it was measured (value at base line, 28.7 ±0.95 seconds; value one hour after heparin, 28.3 ±1.04 seconds; and value three hours after heparin, 27.7 ±0.95 seconds).
Heart Rate and Ventilatory Function
In all 12 subjects the heart rate, respiratory rate, tidal volume, and minute ventilation during rest and exercise were similar regardless of whether the subjects received placebo, cromolyn, or heparin (Table 2). The workload on three experiment days was kept constant for each subject (mean ±SE, 9.6 ±0.9 MET [metabolic equivalents of oxygen consumption]; range, 5.2 to 14.7). During exercise, the heart rate was 162 ±3.6 beats per minute with placebo, 160 ±4.9 beats per minute with cromolyn, and 167 ±4.7 beats per minute with heparin; the minute ventilation was 59.1 ±6.5 liters per minute, 61.0 ±5.7 liters per minute, and 60.3 ±6.8 liters per minute with placebo, cromolyn, and heparin, respectively.
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Post-exercise decreases in SGaw were reproducible in the control and placebo studies; the maximal decrease occurred five minutes after exercise (Figure 1). The maximal decreases in the mean SGaw were 37 ±2 percent and 35 ±2 percent in the control and placebo studies, respectively. Pretreatment with heparin prevented such exercise-induced decreases (Figure 2 and Figure 3), and post-exercise values were not significantly different from base-line values. The maximal decrease in SGaw after pretreatment with heparin was 9 ±5 percent, which was significantly smaller than the decrease after pretreatment with placebo (P<0.05) or cromolyn (P<0.05). Cromolyn partly attenuated post-exercise decreases in SGaw; the maximal decrease was 22 ±5 percent, which was significantly different from the decrease with placebo (P<0.05) (Figure 2 and Figure 3). Heparin completely inhibited the decrease in SGaw (<10 percent) in nine subjects and was ineffective in three subjects. In contrast, cromolyn was effective in three subjects, partly effective in five (SGaw decreases between 10 percent and 25 percent), and ineffective in four (Figure 3).
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Heparin had no effect on histamine-induced bronchoconstriction. The PD values for histamine were 6 ±1.7 breath units after placebo and 4 ±1.4 breath units after heparin (no significant difference).
Discussion
This study demonstrates that inhaled heparin prevents post-exercise bronchoconstriction in subjects with a history of exercise-induced asthma. Heparin had no effect on histamine-induced bronchoconstriction, suggesting that the inhibitory action of heparin may be related to the prevention of mediator release rather than direct effects on smooth muscle. The commercial heparin used in this study contained benzyl alcohol as a preservative. This preservative, when present in high concentration, has been reported to cause relaxation of precontracted canine airway smooth muscle in vitro30. However, it is highly unlikely that the inhibition of exercise-induced asthma by heparin in our study was related to the benzyl alcohol, since the bronchoconstriction was not attenuated by the placebo solution (vehicle alone), which contained an equivalent amount of this preservative.
Various pharmacologic agents, including beta-agonists, cromolyn sodium, atropine, furosemide, and calcium-channel blockers, offer varying degrees of protection against exercise-induced asthma or asthma due to hyperventilation with cold, dry air8,31,32,33. In the present study, both cromolyn sodium and heparin prevented post-exercise bronchoconstriction, but in the dosages used there were significant differences in the efficacy of the two agents. With an equivalent workload and minute ventilation during exercise, the mean SGaw decreased by only 9 percent after pretreatment with heparin (as compared with 35 percent with placebo), thus inhibiting post-exercise bronchoconstriction by 74 percent. In contrast, pretreatment with cromolyn inhibited the response by only 37 percent.
Mast-cell mediators, including histamine and leukotrienes, have been implicated in the pathogenesis of exercise-induced asthma, and H-histamine-receptor antagonists as well as inhibitors of leukotriene D-receptor or 5-lipoxygenase partly attenuate bronchoconstrictor responses induced by exercise or cold, dry air31,34,35. However, the results of the present study in subjects with exercise-induced asthma and previous studies in sheep17,20 indicate that inhaled heparin does not act as an antagonist of mediator receptors. It has been suggested that aerosolized heparin may be helpful in alleviating symptoms of asthma, although no definitive bronchodilating activity has been observed36; this is consistent with our data and suggests that heparin has no direct effect on airway smooth muscle17,19.
Heparin may have multiple sites of action, and previous studies have suggested that antiallergic actions of heparin may be related to the inhibition of mediator release from mast cells17,18,19,20. Heparin was shown to protect against the lethal effects of compound 48/80, which releases histamine, and to prevent mast-cell degranulation induced by this compound in the subcutaneous tissue of mice37. Heparin also attenuated the effect of compound 48/80 and antigen-induced acute bronchoconstrictor responses in sheep, without modifying the effects of histamine17,20. The inhibitory effects of heparin were not related to the alcohol preservative or its anionic charge, and an N-desulfated heparin failed to prevent antigen-induced bronchoconstriction, thus demonstrating the specificity of the action of heparin17. Incubation of a known histamine standard with heparin in vitro had no effect on assay of the standard, and heparin selectively inhibited the anti-IgE-induced release of histamine from human uterine mast cells without modifying the effects of calcium ionophore A2318720. Those observations suggested that heparin attenuated allergic airway responses by modulating the release of mast-cell mediators, rather than by binding, inactivating, or blocking the end-organ effects of the released mediators.
In the present study the interval between pretreatment with heparin and exercise challenge was 45 minutes, an interval similar to that reflecting the pharmacodynamics of the antiallergic action of heparin18. By increasing the preincubation period of heparin in vitro from 20 minutes to 1 hour, the protective effect of heparin on anti-IgE-mediated histamine release and mast-cell degranulation was increased optimally, by 50 percent18. During incubation, heparin may bind to mast-cell membranes and may then become internalized; an active uptake of intratracheally administered heparin by mast cells has been observed38. The proposed pharmacokinetic properties of heparin are consistent with the kinetics of its binding and internalization in other types of cells and indicate a possible intracellular (rather than cell surface) site of action39,40,41.
The antiasthmatic activity of heparin that we observed is probably not related to its anticoagulant properties, since this agent did not prolong the partial-thromboplastin time measured one hour or three hours after inhalation. The administration of an equivalent dose of heparin (1000 U per kilogram) to sheep failed to change the partial-thromboplastin time for up to 12 hours after inhalation17,18.
The mechanism by which heparin attenuates exercise-induced asthma is not clear. The glycosaminoglycan heparin is a mixture of polymers and a linear anionic polyelectrolyte that may act as a pharmacologic mediator42. The heparin molecule possesses multiple non-anticoagulant properties, which include modulation of various proteases, anticomplementary activity, and antiinflammatory action, as well as inhibition of cell growth22,40,41,42,43,44. Heparin has been shown in vitro to bind to inositol triphosphate receptors and to inhibit the inositol triphosphate-induced release of calcium in various tissues, including vascular and airway smooth muscle, the cerebellum, and the liver13,14,15,16. Our observations suggest that the protective effect of heparin against exercise-induced asthma may be related to the inhibition of inositol triphosphate-dependent stimulus-secretion coupling in mast cells. Thus, heparin could be a useful pharmacologic probe in the study of the pathogenesis of exercise-induced asthma and may form a basis of novel therapeutic approaches.
We are indebted to Marvin A. Sackner, M.D., for his help in measuring minute ventilation with the respiratory inductive plethysmograph, and to E. Regis McFadden, Jr., M.D., and Adam Wanner, M.D., for their critical review of the manuscript.
Source Information
From the Division of Pulmonary Disease, University of Miami School of Medicine, Miami, and Mount Sinai Medical Center, Miami Beach, Fla.
Address reprint requests to Dr. Ahmed at the Division of Pulmonary Disease, Mount Sinai Medical Center, 4300 Alton Rd., Miami Beach, FL 33140.
References
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