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Methacholine and physostigmine airway reactivity in asthmatic and nonasthmatic subjects
Methacholine and physostigmine airway reactivity in asthmatic and nonasthmatic subjects Michael
M. Miller,
Chicago,
Ill.
M.D., James E. Fish, M.D.,* and Roy Patterson,
M.D.
Inhalation challenges using methacholine and physostigmine were performed in 3 human asthmatic and 3 nonallergic normal subjects. Plethysmographic measurements of specific airways conductance (GawlVtg) were used to monitor the response. The dose required to produce a 17% fall in GawlVtg was signi&antly lower in asthmatic subjects than in normal subjects for both physostigmine (p < 0.012s) and methacholine (p < 0.05). Moreover, in all subjects the relative airway sensitivity to methacholine correlated with the relative airway sensitivity to physostigmine. Both methacholine and physostigmine are cholinergic agents. Whereas methacholine acts directly at the end organ cholinergic receptor, physostigmine acts by increasing release and decreasing destruction of endogenous acetycholine at the vagal distal innervation. This suggests that the cholinergic airway hyperreactivity characteristic of asthma is a manifestation of end organ hypersensitivity
Bronchial hyperreactivity to the acetylcholine analogue, methacholine, is a characteristic feature of human asthma.le4 Similar to human asthmatics, rhesus monkeys with immediate-type, reagin-mediated asthma following aerosol challenge with Ascaris suum antigen also demonstrate bronchial hypet-reactivity to aerosolized carbachol, another acetylcholine analogue. 5, 6 However, asthmatic monkeys do not demonstrate bronchial hyperreactivity when they are challenged with physostigmine sulfate, an anticholinesterase agent that stimulates release and inhibits destruction of endogenous acetylcholine at the efferent parasympathetic nerve terminal.5 The present study was undertaken to determine whether human asthmatics lack bronchial hyperreactivity to endogenous acetylcholine released locally in
From the Allergy-Immunology and Pulmonary Disease Sections, Department of Medicine, Northwestern University Medical School. Supported by the Ernest S. Bazley Grant, United States Public Health Service Grant AI 11759, and the Chicago Lung Association. Received for publication March 2, 1977. Accepted for publication May 24, 1977. Reprint requests to: Roy Patterson, M.D., Section of AllergyImmunology, Northwestern University Medical School, 303 East Chicago Ave., Chicago, Ill. 60611. *Recipient of the Edward L. Trudeau Fellowship Award of the American Lung Association, and the Pulmonary Young Investigator Award of the National Heart and Lung Institute (HL 19577-01). Vol. 60, No. 2, pp. 116-120
vivo by aerosol challenge with physostigmine and to compare methacholine and physostigmine responses in asthmatic and nonasthmatic subjects. METHODS After obtaining informed consent, inhalation doseresponse curves for methacholine and physostigmine were established in three nonallergic normal control subjects and in three subjects with allergic asthma. Table I lists the baseline characteristics of these subjects. Only volunteer physicians at this institution who were fully cognizant of the known side effects and potential toxicity of physostigmine were chosen for the study. All subjects were between 30 and 35 years of age, and only Subject I was a cigarette smoker. Subject 4 had seasonal allergic rhinitis and very mild allergic asthma precipitated only by cat dander; he did not have chronic asthma and did not require regular bronchodilator therapy. Subject 5 had mild allergic asthma without allergic rhinitis. His symptoms were precipitated by severe exercise and animal dander exposure, rarely requiring bronchodilators. Subject 6 had seasonal allergic rhinitis and chronic asthma requiring regular oral bronchodilators. No subject
was taking
corticosteroids
at the time of the
study, and all bronchodilators were withheld for sixteen hours before the challenge. The methacholine challenge was performed first and the physostigmine challenge was performed one week later. Both challenge agents were administered in increasing concentrations and were delivered to the lung through a DeVilbiss No. 42 nebulizer. The nebulizer was attached to a dose-metering device, which consisted of a breathactivated solenoid valve, timing circuit, and a source of
compressed air under 20 psi pressure. The solenoid valve
VOLUME NUMBER
TABLE
Methacholine
60 2
I. Haseline
pulmonary
function
before
Methacholine
Subjects
Normal I 2 3 Asthmarrc 4 5 6
methacholine
airway
5.55 (4.75) 4.70 (4.55) 6.30 (5.50)
77 80 81
0.228 0.166 0.150
4.90 (4.99) 4.52 (4.82) 5.22 (4.50)
85 67 75
0.165 0.099 0.156
reactivity
baseline
FEV, %, Fvc
GawlVtg (set+ cm Hz0 ‘1
5.64 4.91 6.27
79 74 81
0.154 0.193 0.153
5.10 4.69 4.97
84 68 68
0.159 0.141 0.108
Fw
(Lb
117
challenge
Physostigmine GawlVtg (set ‘cm H,O-‘ f
(L)
physostigmine
baseline
FEV, %, Fvc
FVC (Pred*)
and
and physostigmine
*Predicted values from Morris, J. F., et al.” was set to remain open for 0.6 set during inhalation, allowing the compressed air to flow through the nebulizer and dispersing an average of 0.023 cc of the solution with each breath. The various concentrations of physostigmine and methacholine were prepared from a 25mglml stock solution using 0.9% phosphate-buffered saline as the diluent. Each of the two agents was administered according to the following protocol. Each subject took five inhalations of a control aerosol containing the buffered saline diluent alone. We then established dose-response curves by having the subject take five breaths each of successively increasing concentrations of the agent administered at five-minute intervals. The initial concentrations used were 0.078 mg/ml for methacholine and 0.19 mgiml for physostigmine; subsequent concentrations were increased in twofold increments. Subject 6 was given an initial physostigmine concentration of 0.095 mg/ml because of his inordinate sensitivity to methacholine. Because of the potential systemic response to physostigmine, each subject rinsed his mouth with tap water after each dose of physostigmine to minimize absorption through the oral and gastrointestinal mucosae. Baseline measurements of forced expiration and specific airways conductance (Gaw/Vtg) were obtained prior to each day’s testing. Forced expiration was performed using an automated “Wedge” spirometer (Model 270, MedScience Electronics, St. Louis, Missouri), and measurements of the forced vital capacity (FVC) and the onesecond forced expired volume (FEV,) were made. Specific airways conductance was determined in the following manner. Airwa:y resistance was determined by the methods of Dubois and co-workers’, x using a variable-pressure body plethysmograph and converted to Gaw/Vtg by dividing the reciprocal of the resistance by the thoracic gas volume at which the measurement was made. Three measurements of Gaw/Vtg were obtained following the control aerosol and during the five-minute interval between doses of the challenge agenls, and the average of the three measurements was used. Blood pressure, pulse rate, and pupil size were also measured to monitor systemic effects of the drugs. The inhalation challenge was terminated when Gaw/Vtg fell 50% from the saline aerosol control value or when the subjects experienced moderate respiratory or systemic symptoms. Atropine sulfate (0.6 mg) was given subcutaneously
after the challenge to any subject experiencing these symptoms. Dose-response curves were constructed by plotting the change in Gaw/Vtg, expressed as a percent of the aerosol control value, against the cumulative dose of physostigmine or methacholine given. Data were analyzed using the rank correlation coefficient and Student’s t distribution for group mean differences.Y
RESULTS Baseline values of the FVC and FEV, expressed as a percent of the FVC (FEV,%, FVC) and Gaw/Vtg prior to aerosol challenge are shown in Table I. Although two of the three asthmatic subjects demonstrated mild airway obstruction prior to testing, the baseline values on the two test days were comparable for individual subjects. Fig. 1 depicts the dose-response relationship to methacholine for each subject. The cumulative provocation dose of methacholine producing a 17% drop in Gaw/Vtg (PD,, Gaw/Vtg) was used to compare bronchial sensitivity. The PD1, Gaw/Vtg was chosen for comparison because this was the maximum fall achieved by Subject 1 during the physostigmine challenge; hence this was the largest common fall at which the bronchial sensitivities to methacholine and physostigmine could be compared for all subjects. In response to methacholine the asthmatic subjects demonstrated significantly greater bronchial reactivity than normals. The mean *SD PD,, Gaw/Vtg for the asthmatic and normal subjects was 0.014 rt 0.01 mg and 1.46 +- 0.88 mg, respectively (p < 0.05). The dose-response relationship for physostigmine in the asthmatic and normal subjects is illustrated in Fig. 2. The asthmatic subjects were also more sensitive to physotigmine than the normal subjects. The mean +- SD PD,, Gaw/Vtg was 0.055 -+:0.06 mg for asthmatic subjects and 2.08 -C 0.81 mg for normal subjects (p < 0.0125). Subjects 5 and 6 received cumulative doses of
118
Miller,
Fish, and Patterson
J. ALLERGY
Cumulative
Dose of Methacholine
(mg)
FIG. 1. Methacholine dose-response relationship in 3 normal (----) and 3 asthmatic Methacholine dose is expressed as the cumulative amount of methacholine in milligrams challenge. GawlVtg is expressed as the percent of the saline aerosol control value. Numbers individual subjects (see Table I).
Cumulative
Dose of
Physostigmine
Sulfate
(-1 subjects. given during the in circles refer to
(mg)
FIG. 2. Physostigmine dose-response relationship in normal (----) and asthmatic (-1 mine dose represents the cumulative amount in milligrams given during the challenge. as the percent of the saline aerosol control value. Numbers in circles refer to individual
physostigmine that were less than 0.065 mg and experienced no systemic effects. Subjects 1, 2, 3, and 4 received cumulative doses of physostigmine in excess of 0.7 mg and experienced lightheadedness, eructation, nausea, and diaphoresis. Tremors, distal paresthesias, tenesmus, and a 25% reduction in pulse rate also occurred in Subject 1. No changes in blood pressure or pupil size were observed. Using the PD17 Gaw/Vtg for comparison, in all six subjects the relative bronchial reactivity to
CLIN. IMMUNOL. AUGUST 1977
subjects. PhysostigGawfVtg is expressed subjects (see Table I).
methacholine correlates with the relative bronchial reactivity to physostigmine (r, = 1, p < 0.01). When analyzed separately, the small size of the asthmatic and normal groups precludes determination of any significant rank correlation between methacholine and physostigmine sensitivity. However, within these two subgroups the subjects possessing the greatest and least degree of bronchial reactivity to methacholine also possess the greatest and least degree of bronchial reactivity to physostigmine.
VOLUME NUMBER
60 2
DISCUSSION Because of the untoward systemic effects of physostigmine that were observed during these experiments, we chose not to extend our observations by challenging additional subjects. Despite the small number of subjects tested, these studies demonstrate significantly greater bronchial reactivity to both methacholine and physostigmine in asthmatic subjects compared to normal subjects. In both normal and asthmatic subjects the relative airway sensitivity to physostigmine correlated with the relative sensitivity to methacholine. Bronchial hyperreactivity to aerosolized methacholine in asthmatic subjects has been well established.lm4 In addition, asthmatic subjects also demonstrate bronchial hyperreactivity to inhaled irritants such as sulfur dioxide, smoke, and citric acid. The bronchoconstriction produced by inhaled irritants can be prevented by prior administration of atropine, and therefore the response is thought to be mediated by a vagal parasympathetic reflex initiated by stimulation of epithelial irritant receptors located in the airways. lo It has been suggested that this exaggerated airway reactivity characteristic of asthma is a result of either end organ cholinergic hypersensitivity or an increase in irritant receptor sensitivity.” Whereas methacholine produces cholinergic stimulation by its direct pharmacologic effect on the cholinergic recepter, physostigmine acts by stimulating release of endogenous acetylcholine from the postganglionic parasympathetic nerve endings and by inhibiting the destruction of acetylcholine.12-i4 In the absence of a functional nervous system, physostigmine produces no cholinergic stimulation. This has been demonstrated by its loss of cholinergic activity following neuronal blockade with local anesthetics or after anatomic denervation of the end organ.12, 15*I6 In contrast:, the end organ response produced by methacholine is not inhibited by pharmacologic or anatomic denervation.is, i6 We have shown that asthmatic subjects demonstrate bronchial hyperreactivity to physotigmineinduced release of acetycholine from the distal efferent postganglionic parasympathetic nerve terminal as well as to the direct pharmacologic action of methacholine. Although these studies do not rule out the possibility of irritant receptor hypersensitivity, the greater responsiveness of asthmatic subjects to physostigmine compared to normals suggests that the cholinergic airway hyperreactivity characteristic of asthma is more likely a function of end organ hypersensitivity than a function of excessive vagal stimulation. In contra.st to the responses we observed in human
Methacholine
and physostigmine
airway
reactivity
119
subjects, Miller and Patterson5 failed to demonstrate bronchial hyperreactivity to physostigmine in rhesus monkeys with reagin-mediated asthma, although the same monkeys were more sensitive to the acetylcholine analogue, carbachol, than nonasthmatic monkeys. Attempts to explain the discrepancy between the human and monkey responses are important since rhesus monkeys possessing immediate-type reaginmediated asthma have become a useful model for the study of human asthma. Although it is possible that a species difference could account for the differences in physostigmine reactivity, we feel that this is unlikely because both species possess cholinergic hyperreactivity to acetylcholine analogues and there is no evidence in support of a species difference in the postganglionic efferent parasympathetic innervation of the lung. Another possible explanation for the discrepancy is the unknown potential effect of anesthesia on either the physiologic response to inhalation challenge or to its effect on the release of endogenous acetylcholine from parasympathetic nerve terminals. Whereas monkeys were studied under general anesthesia, the human subjects in this study had no anesthetic suppression. REFERENCES I. Fish, J. E., Rosenthal, R. R., Batra, G., Menkes, H., Summer, W., Permutt, S., Norman, P.: Airway responses to methacholine in allergic and nonallergic subjects, Am. Rev. Respir. Dis. 113:579, 1976. 2. Parker, C. C., Bilbo, R. E., and Reed, C. E.: Methacholine aerosol as a test for bronchial asthma, Arch. Intern. Med. 115:452, 1965. 3. Spector, S. L., and Farr, R. S.: A comparison of methacholine and histamine inhalations in asthmatics, J. ALLERGY CLIN. IMMUNOL. 56308, 1975. 4. Townley, R. Cl., Ryo, U. Y., Kolotkin, B. M., and Kang, B.: Bronchial sensitivity to methacholine in current and former asthmatic and allergic rhinitis patients and control subjects, J. ALLERCYCLIN.IMMUNOL. 56~429, 1975. 5. Miller, M. M., and Patterson, R.: Differential airway reactivity to carbachol and physostigmine sulfate in rhesus monkeys with and without reagin-mediated respiratory responses, Int. Arch. Allergy Appl. Immunol. 53:349, 1977. 6. Patterson, R., Harris, K. E., Suszko, I. M., and Roberts, M.: Reagin-mediated asthma in rhesus monkeys and relation to bronchial cell histamine release and airway reactivity to carbocholine, J. Clin. Invest. 57:586, 1976. 7. DuBois, A. B., Botelho, S. Y., and Comroe, J. H., Jr.: A new method for measuring airway resistance in man using a body plethysmograph: Values in normal subjects and in patients with respiratory disease, J. Clin. Invest. 35:327, 1956. 8. DuBois, A. B., Botelho, S. Y., Bedell, G. N., Marshall, R., and Comroe, I. H., Jr.: A rapid plethysmographic method for measuring thoracic gas: A comparison with a nitrogen washout method for measuring functional residual capacity in normal subjects, J. Clin. Invest. 35:327, 1956.
120
Miller,
Fish, and Patterson
9. Snedecor, G. W., and Cochran, W. G.: Statistical methods, Ames, Iowa, 1967, Iowa State University Press. 10. Simonsson, B. G., Jacobs, F. M., and Nadel, J. A.: Role of autonomic nervous system and the cough reflex in the increased responsiveness of airways patients with obstructive airway disease, J. Clin. Invest. 46:1812, 1967. 11. Nadel, J. A.: Neurophysiologic Aspects of Asthma, in Austen, K. F., and Lichtenstein, L. M., editors: Asthma, New York, 1973, Academic Press, Inc., pp. 29-37. 12. Bell, C.: Effects of physostigmine on smooth muscle, Biochem. Pharmacol. 15:1085, 1966. 13. Carlyle, R. F.: The mode of action of neostigmine and physostigmine on the guinea pig trachealis muscle, Br. J. pharmacol. 21:137, 1963.
J. ALLERGY
CLIN. IMMUNOL. AUGUST 1977
14. Cox, B.: Effects of two cholinesterase inhibitors on acetylcholine release from the guinea pig isolated ilium preparation, J. Pharmacol. 22~231, 1970. 15. Goodman, L. S., and Gilman, A.: Pharmacologic basis of therapeutics, New York, 1970, MacMillan Publishing Co., Inc. 16. Miller, M. M., Patterson, R., and Harris, K. E.: A comparison of immunologic asthma to two types of cholinergic respiratory responses in the rhesus monkey, J. Lab. Clin. Med. 88~995, 1976. 17. Morris, J. F., Koski, A., and Johnson, L. C.: Spirometric standards for healthy non-smoking adults, Am. Rev. Respir. Dis. 103~57, 1971.
M. Miller,
Chicago,
Ill.
M.D., James E. Fish, M.D.,* and Roy Patterson,
M.D.
Inhalation challenges using methacholine and physostigmine were performed in 3 human asthmatic and 3 nonallergic normal subjects. Plethysmographic measurements of specific airways conductance (GawlVtg) were used to monitor the response. The dose required to produce a 17% fall in GawlVtg was signi&antly lower in asthmatic subjects than in normal subjects for both physostigmine (p < 0.012s) and methacholine (p < 0.05). Moreover, in all subjects the relative airway sensitivity to methacholine correlated with the relative airway sensitivity to physostigmine. Both methacholine and physostigmine are cholinergic agents. Whereas methacholine acts directly at the end organ cholinergic receptor, physostigmine acts by increasing release and decreasing destruction of endogenous acetycholine at the vagal distal innervation. This suggests that the cholinergic airway hyperreactivity characteristic of asthma is a manifestation of end organ hypersensitivity
Bronchial hyperreactivity to the acetylcholine analogue, methacholine, is a characteristic feature of human asthma.le4 Similar to human asthmatics, rhesus monkeys with immediate-type, reagin-mediated asthma following aerosol challenge with Ascaris suum antigen also demonstrate bronchial hypet-reactivity to aerosolized carbachol, another acetylcholine analogue. 5, 6 However, asthmatic monkeys do not demonstrate bronchial hyperreactivity when they are challenged with physostigmine sulfate, an anticholinesterase agent that stimulates release and inhibits destruction of endogenous acetylcholine at the efferent parasympathetic nerve terminal.5 The present study was undertaken to determine whether human asthmatics lack bronchial hyperreactivity to endogenous acetylcholine released locally in
From the Allergy-Immunology and Pulmonary Disease Sections, Department of Medicine, Northwestern University Medical School. Supported by the Ernest S. Bazley Grant, United States Public Health Service Grant AI 11759, and the Chicago Lung Association. Received for publication March 2, 1977. Accepted for publication May 24, 1977. Reprint requests to: Roy Patterson, M.D., Section of AllergyImmunology, Northwestern University Medical School, 303 East Chicago Ave., Chicago, Ill. 60611. *Recipient of the Edward L. Trudeau Fellowship Award of the American Lung Association, and the Pulmonary Young Investigator Award of the National Heart and Lung Institute (HL 19577-01). Vol. 60, No. 2, pp. 116-120
vivo by aerosol challenge with physostigmine and to compare methacholine and physostigmine responses in asthmatic and nonasthmatic subjects. METHODS After obtaining informed consent, inhalation doseresponse curves for methacholine and physostigmine were established in three nonallergic normal control subjects and in three subjects with allergic asthma. Table I lists the baseline characteristics of these subjects. Only volunteer physicians at this institution who were fully cognizant of the known side effects and potential toxicity of physostigmine were chosen for the study. All subjects were between 30 and 35 years of age, and only Subject I was a cigarette smoker. Subject 4 had seasonal allergic rhinitis and very mild allergic asthma precipitated only by cat dander; he did not have chronic asthma and did not require regular bronchodilator therapy. Subject 5 had mild allergic asthma without allergic rhinitis. His symptoms were precipitated by severe exercise and animal dander exposure, rarely requiring bronchodilators. Subject 6 had seasonal allergic rhinitis and chronic asthma requiring regular oral bronchodilators. No subject
was taking
corticosteroids
at the time of the
study, and all bronchodilators were withheld for sixteen hours before the challenge. The methacholine challenge was performed first and the physostigmine challenge was performed one week later. Both challenge agents were administered in increasing concentrations and were delivered to the lung through a DeVilbiss No. 42 nebulizer. The nebulizer was attached to a dose-metering device, which consisted of a breathactivated solenoid valve, timing circuit, and a source of
compressed air under 20 psi pressure. The solenoid valve
VOLUME NUMBER
TABLE
Methacholine
60 2
I. Haseline
pulmonary
function
before
Methacholine
Subjects
Normal I 2 3 Asthmarrc 4 5 6
methacholine
airway
5.55 (4.75) 4.70 (4.55) 6.30 (5.50)
77 80 81
0.228 0.166 0.150
4.90 (4.99) 4.52 (4.82) 5.22 (4.50)
85 67 75
0.165 0.099 0.156
reactivity
baseline
FEV, %, Fvc
GawlVtg (set+ cm Hz0 ‘1
5.64 4.91 6.27
79 74 81
0.154 0.193 0.153
5.10 4.69 4.97
84 68 68
0.159 0.141 0.108
Fw
(Lb
117
challenge
Physostigmine GawlVtg (set ‘cm H,O-‘ f
(L)
physostigmine
baseline
FEV, %, Fvc
FVC (Pred*)
and
and physostigmine
*Predicted values from Morris, J. F., et al.” was set to remain open for 0.6 set during inhalation, allowing the compressed air to flow through the nebulizer and dispersing an average of 0.023 cc of the solution with each breath. The various concentrations of physostigmine and methacholine were prepared from a 25mglml stock solution using 0.9% phosphate-buffered saline as the diluent. Each of the two agents was administered according to the following protocol. Each subject took five inhalations of a control aerosol containing the buffered saline diluent alone. We then established dose-response curves by having the subject take five breaths each of successively increasing concentrations of the agent administered at five-minute intervals. The initial concentrations used were 0.078 mg/ml for methacholine and 0.19 mgiml for physostigmine; subsequent concentrations were increased in twofold increments. Subject 6 was given an initial physostigmine concentration of 0.095 mg/ml because of his inordinate sensitivity to methacholine. Because of the potential systemic response to physostigmine, each subject rinsed his mouth with tap water after each dose of physostigmine to minimize absorption through the oral and gastrointestinal mucosae. Baseline measurements of forced expiration and specific airways conductance (Gaw/Vtg) were obtained prior to each day’s testing. Forced expiration was performed using an automated “Wedge” spirometer (Model 270, MedScience Electronics, St. Louis, Missouri), and measurements of the forced vital capacity (FVC) and the onesecond forced expired volume (FEV,) were made. Specific airways conductance was determined in the following manner. Airwa:y resistance was determined by the methods of Dubois and co-workers’, x using a variable-pressure body plethysmograph and converted to Gaw/Vtg by dividing the reciprocal of the resistance by the thoracic gas volume at which the measurement was made. Three measurements of Gaw/Vtg were obtained following the control aerosol and during the five-minute interval between doses of the challenge agenls, and the average of the three measurements was used. Blood pressure, pulse rate, and pupil size were also measured to monitor systemic effects of the drugs. The inhalation challenge was terminated when Gaw/Vtg fell 50% from the saline aerosol control value or when the subjects experienced moderate respiratory or systemic symptoms. Atropine sulfate (0.6 mg) was given subcutaneously
after the challenge to any subject experiencing these symptoms. Dose-response curves were constructed by plotting the change in Gaw/Vtg, expressed as a percent of the aerosol control value, against the cumulative dose of physostigmine or methacholine given. Data were analyzed using the rank correlation coefficient and Student’s t distribution for group mean differences.Y
RESULTS Baseline values of the FVC and FEV, expressed as a percent of the FVC (FEV,%, FVC) and Gaw/Vtg prior to aerosol challenge are shown in Table I. Although two of the three asthmatic subjects demonstrated mild airway obstruction prior to testing, the baseline values on the two test days were comparable for individual subjects. Fig. 1 depicts the dose-response relationship to methacholine for each subject. The cumulative provocation dose of methacholine producing a 17% drop in Gaw/Vtg (PD,, Gaw/Vtg) was used to compare bronchial sensitivity. The PD1, Gaw/Vtg was chosen for comparison because this was the maximum fall achieved by Subject 1 during the physostigmine challenge; hence this was the largest common fall at which the bronchial sensitivities to methacholine and physostigmine could be compared for all subjects. In response to methacholine the asthmatic subjects demonstrated significantly greater bronchial reactivity than normals. The mean *SD PD,, Gaw/Vtg for the asthmatic and normal subjects was 0.014 rt 0.01 mg and 1.46 +- 0.88 mg, respectively (p < 0.05). The dose-response relationship for physostigmine in the asthmatic and normal subjects is illustrated in Fig. 2. The asthmatic subjects were also more sensitive to physotigmine than the normal subjects. The mean +- SD PD,, Gaw/Vtg was 0.055 -+:0.06 mg for asthmatic subjects and 2.08 -C 0.81 mg for normal subjects (p < 0.0125). Subjects 5 and 6 received cumulative doses of
118
Miller,
Fish, and Patterson
J. ALLERGY
Cumulative
Dose of Methacholine
(mg)
FIG. 1. Methacholine dose-response relationship in 3 normal (----) and 3 asthmatic Methacholine dose is expressed as the cumulative amount of methacholine in milligrams challenge. GawlVtg is expressed as the percent of the saline aerosol control value. Numbers individual subjects (see Table I).
Cumulative
Dose of
Physostigmine
Sulfate
(-1 subjects. given during the in circles refer to
(mg)
FIG. 2. Physostigmine dose-response relationship in normal (----) and asthmatic (-1 mine dose represents the cumulative amount in milligrams given during the challenge. as the percent of the saline aerosol control value. Numbers in circles refer to individual
physostigmine that were less than 0.065 mg and experienced no systemic effects. Subjects 1, 2, 3, and 4 received cumulative doses of physostigmine in excess of 0.7 mg and experienced lightheadedness, eructation, nausea, and diaphoresis. Tremors, distal paresthesias, tenesmus, and a 25% reduction in pulse rate also occurred in Subject 1. No changes in blood pressure or pupil size were observed. Using the PD17 Gaw/Vtg for comparison, in all six subjects the relative bronchial reactivity to
CLIN. IMMUNOL. AUGUST 1977
subjects. PhysostigGawfVtg is expressed subjects (see Table I).
methacholine correlates with the relative bronchial reactivity to physostigmine (r, = 1, p < 0.01). When analyzed separately, the small size of the asthmatic and normal groups precludes determination of any significant rank correlation between methacholine and physostigmine sensitivity. However, within these two subgroups the subjects possessing the greatest and least degree of bronchial reactivity to methacholine also possess the greatest and least degree of bronchial reactivity to physostigmine.
VOLUME NUMBER
60 2
DISCUSSION Because of the untoward systemic effects of physostigmine that were observed during these experiments, we chose not to extend our observations by challenging additional subjects. Despite the small number of subjects tested, these studies demonstrate significantly greater bronchial reactivity to both methacholine and physostigmine in asthmatic subjects compared to normal subjects. In both normal and asthmatic subjects the relative airway sensitivity to physostigmine correlated with the relative sensitivity to methacholine. Bronchial hyperreactivity to aerosolized methacholine in asthmatic subjects has been well established.lm4 In addition, asthmatic subjects also demonstrate bronchial hyperreactivity to inhaled irritants such as sulfur dioxide, smoke, and citric acid. The bronchoconstriction produced by inhaled irritants can be prevented by prior administration of atropine, and therefore the response is thought to be mediated by a vagal parasympathetic reflex initiated by stimulation of epithelial irritant receptors located in the airways. lo It has been suggested that this exaggerated airway reactivity characteristic of asthma is a result of either end organ cholinergic hypersensitivity or an increase in irritant receptor sensitivity.” Whereas methacholine produces cholinergic stimulation by its direct pharmacologic effect on the cholinergic recepter, physostigmine acts by stimulating release of endogenous acetylcholine from the postganglionic parasympathetic nerve endings and by inhibiting the destruction of acetylcholine.12-i4 In the absence of a functional nervous system, physostigmine produces no cholinergic stimulation. This has been demonstrated by its loss of cholinergic activity following neuronal blockade with local anesthetics or after anatomic denervation of the end organ.12, 15*I6 In contrast:, the end organ response produced by methacholine is not inhibited by pharmacologic or anatomic denervation.is, i6 We have shown that asthmatic subjects demonstrate bronchial hyperreactivity to physotigmineinduced release of acetycholine from the distal efferent postganglionic parasympathetic nerve terminal as well as to the direct pharmacologic action of methacholine. Although these studies do not rule out the possibility of irritant receptor hypersensitivity, the greater responsiveness of asthmatic subjects to physostigmine compared to normals suggests that the cholinergic airway hyperreactivity characteristic of asthma is more likely a function of end organ hypersensitivity than a function of excessive vagal stimulation. In contra.st to the responses we observed in human
Methacholine
and physostigmine
airway
reactivity
119
subjects, Miller and Patterson5 failed to demonstrate bronchial hyperreactivity to physostigmine in rhesus monkeys with reagin-mediated asthma, although the same monkeys were more sensitive to the acetylcholine analogue, carbachol, than nonasthmatic monkeys. Attempts to explain the discrepancy between the human and monkey responses are important since rhesus monkeys possessing immediate-type reaginmediated asthma have become a useful model for the study of human asthma. Although it is possible that a species difference could account for the differences in physostigmine reactivity, we feel that this is unlikely because both species possess cholinergic hyperreactivity to acetylcholine analogues and there is no evidence in support of a species difference in the postganglionic efferent parasympathetic innervation of the lung. Another possible explanation for the discrepancy is the unknown potential effect of anesthesia on either the physiologic response to inhalation challenge or to its effect on the release of endogenous acetylcholine from parasympathetic nerve terminals. Whereas monkeys were studied under general anesthesia, the human subjects in this study had no anesthetic suppression. REFERENCES I. Fish, J. E., Rosenthal, R. R., Batra, G., Menkes, H., Summer, W., Permutt, S., Norman, P.: Airway responses to methacholine in allergic and nonallergic subjects, Am. Rev. Respir. Dis. 113:579, 1976. 2. Parker, C. C., Bilbo, R. E., and Reed, C. E.: Methacholine aerosol as a test for bronchial asthma, Arch. Intern. Med. 115:452, 1965. 3. Spector, S. L., and Farr, R. S.: A comparison of methacholine and histamine inhalations in asthmatics, J. ALLERGY CLIN. IMMUNOL. 56308, 1975. 4. Townley, R. Cl., Ryo, U. Y., Kolotkin, B. M., and Kang, B.: Bronchial sensitivity to methacholine in current and former asthmatic and allergic rhinitis patients and control subjects, J. ALLERCYCLIN.IMMUNOL. 56~429, 1975. 5. Miller, M. M., and Patterson, R.: Differential airway reactivity to carbachol and physostigmine sulfate in rhesus monkeys with and without reagin-mediated respiratory responses, Int. Arch. Allergy Appl. Immunol. 53:349, 1977. 6. Patterson, R., Harris, K. E., Suszko, I. M., and Roberts, M.: Reagin-mediated asthma in rhesus monkeys and relation to bronchial cell histamine release and airway reactivity to carbocholine, J. Clin. Invest. 57:586, 1976. 7. DuBois, A. B., Botelho, S. Y., and Comroe, J. H., Jr.: A new method for measuring airway resistance in man using a body plethysmograph: Values in normal subjects and in patients with respiratory disease, J. Clin. Invest. 35:327, 1956. 8. DuBois, A. B., Botelho, S. Y., Bedell, G. N., Marshall, R., and Comroe, I. H., Jr.: A rapid plethysmographic method for measuring thoracic gas: A comparison with a nitrogen washout method for measuring functional residual capacity in normal subjects, J. Clin. Invest. 35:327, 1956.
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9. Snedecor, G. W., and Cochran, W. G.: Statistical methods, Ames, Iowa, 1967, Iowa State University Press. 10. Simonsson, B. G., Jacobs, F. M., and Nadel, J. A.: Role of autonomic nervous system and the cough reflex in the increased responsiveness of airways patients with obstructive airway disease, J. Clin. Invest. 46:1812, 1967. 11. Nadel, J. A.: Neurophysiologic Aspects of Asthma, in Austen, K. F., and Lichtenstein, L. M., editors: Asthma, New York, 1973, Academic Press, Inc., pp. 29-37. 12. Bell, C.: Effects of physostigmine on smooth muscle, Biochem. Pharmacol. 15:1085, 1966. 13. Carlyle, R. F.: The mode of action of neostigmine and physostigmine on the guinea pig trachealis muscle, Br. J. pharmacol. 21:137, 1963.
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14. Cox, B.: Effects of two cholinesterase inhibitors on acetylcholine release from the guinea pig isolated ilium preparation, J. Pharmacol. 22~231, 1970. 15. Goodman, L. S., and Gilman, A.: Pharmacologic basis of therapeutics, New York, 1970, MacMillan Publishing Co., Inc. 16. Miller, M. M., Patterson, R., and Harris, K. E.: A comparison of immunologic asthma to two types of cholinergic respiratory responses in the rhesus monkey, J. Lab. Clin. Med. 88~995, 1976. 17. Morris, J. F., Koski, A., and Johnson, L. C.: Spirometric standards for healthy non-smoking adults, Am. Rev. Respir. Dis. 103~57, 1971.