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In Vivo and in Vitro Studies on Alpha-receptors in Human Airways; Potentiation with Bacterial Endotoxins
35
15TH ANNUAL ASPEN CONFERENCE
sone; 2) systemic corticosteroid administration restores the responsiveness of adenyl cyclase to betaadrenergic stimulation; 3) systemic corticosteroids inhibit the increased ATPase activity, possibly with a concomitant decrease in transport, contractile and secretory processes; and 4) corticosteroids at high levels may inhibit cyclic AMP phosphodiesterase activity. The data also suggest that the therapeutic use of alpha-adrenergic blocking agents such as phentolamine, shown to be effective against asthma in Europe," should be studied clinically.
4
5
6
7 REFERENCES
1 Szentivanyi A: The beta-adrenergic theory of the atopic abnormality in bronchial asthma. J Allergy 42:203, 1968 2 Townley RG, Hanrath T, Guirgis HM : The inhibitory effect of hydrocortisone on the alpha adrenergic responses of human and guinea pig isolated respiratory smooth muscle. J Allergy Clin Immunol 49 :88, 1972 3 Coffey RG, Hadden JW, Hadden EM, et al l Stimulation of
8
9
ATPase by norepinephrine : An alpha-adrenergic receptor mechanism. Fed Proc 30:497, 1971 Bourne HR, Melmon KL: Adenyl cyclase in human leukocytes : eVidence for activation by separate beta-adrenergic and prostaglandin receptors. J Pharm Exper Therap 178: 1, 1971 Logsdon PJ, Middleton E Jr, Coffey RG: Stimulation of leukocyte adenyl cyclase by hydrocortisone and isoproterenol in asthmatic and non-asthmatic subjects. J Allergy Clin Immunol (in press) Logsdon PJ, Camright DV, Middleton E jr, et al : Effects of theophylline and aminophylline on stimulation of leukocyte adenyl cyclase in normal and asthmatic subjects. Biochem Biophys Res Commun (in press) Hadden JW, Hadden EM, Wilson EE, et al l Direct action of insulin on plasma membrane ATPase in human leukocytes. Nature, 235: 174, 1972 Orange RP, Kaliner MA, LaRaia PJ, et al : Immunologic release of histamine and slow reacting substance (Jf anaphylaxis from human lung . Influence of cellular levels of cyclic AMP Fed Proc 30:1725,1971 Marcelle R, Bottin R, Juchmes J, et al : Reactions bronchomotrica des receptors beta-adrenergiques. Acta Allergol 23:11,1968
In vivo and in vitro Studies on Alpha-receptors in Human Airways; Potentiation with Bacterial Endotoxins· Bo G. Simonsson, M.D.; N. Svedmyr, M.D.; B. -E. Skoogh, M.D.;
R. Anderson, M.D.; and N. P. Bergh, M.D.
The increased responsiveness of the airway caliber-regulating system seen in asthmatics may be due to increased sensitivity of the afferent nerve endings, ie the irritant receptors, or of the smooth bronchial muscle receptors. SzentivanyP has proposed a theory of functional beta-receptor blockade as a cause of airway obstruction in disease. A possible alpha-receptor stimulation would then cause bronchoconstriction. Studies in animals have shown the presence of alpha-receptors in the airway muscles; in Croningen in 1969 we gave a preliminary report on the first simultaneous evidence in vivo and in vitro of the presence of bronchoconstricting alphareceptors in human bronchial muscle." We have studied the effect in vivo of cholinergic blockade, adrenergic beta-blockade and alpha stimulation in 15 patients, most with airway ob°From the Department of Pharmacology, the Thoracic Surgery Clinic and the Lung Clinic, Benstromska Siukhuset, University of Coteborg, Sweden. o °Present address : University Lung Clinic ; Lasarettet, S-221 85 Lund, Sweden.
struction. In vitro studies on human bronchial muscles confirm the findings and show potentiation of alpha-receptor bronchoconstriction after treatment with endotoxin. We measured airway conductance (Caw) at different lung volumes in a volume-displacement body plethysmograph and computed conductance lines from which a computer compared Caw at specific subvolumes of the control total lung capacity (20, 55 and 80 percent VC) . We measured conductance during five-minute periods in the control state, ten minutes after IV injection of atropine (cholinergic blockade), 5-15 minutes after IV propranalol (,B-blockade), and 5-10 minutes after inhalation of metaoxedrine, a specific a-receptor stimulator.
Results in vivo Atropine caused bronchodilation in most subjects. Propranolol induced varying responses, the mean Caw was unchanged compared to the study post-
CHEST, VOL. 63, NO.4, APRIL 1973 SUPPLEMENT
45
15TH ANNUAL ASPEN CONFERENCE Q.Q2
i o,) ~f ~
0
•
0
1
.~a PHE
•
I
3min
5''d 1
1.7.ifJ PHE
EHDOTOX.
6 ' Jrnin
J
;
Q.02~ ~O.l ~o
o
3
3
I min 0 • • min 1. The effect of metaoxedrine (PHE) in vitro before and after addition of bacterial endotoxins on a previously not ,a-blocked human bronchial muscle . Below: Increased sensitivity for histamine in the same preparations.
FIGURE
atropine. After blockade of the beta-receptors and the vagal reflex, metaoxedrine induced a decrease of Caw in all but two subjects. The mean Gaw between the study after propranolol and the study after metaoxedrine was 0.17 (I/s)cm H20 (P
Results in vitro The in vitro studies were performed on specimens of segmental bronchi of 3-5 mm diameter obtained from patients undergoing operation for lung tumor. Preparations with a breadth of 5-10 mm and length of about 15 mm were then mounted in plastic holders according to Lundholm and Mohme-Lundholm." Tension changes of the circular muscle layer were measured with a Statham transducer on a Crass polygraph. The muscle was suspended in a buffer solution at 37° C and the various drugs were tested after adequate flushing of the preparations. The alpha-mediated bronchoconstriction was studied after treatment with a lipo-polysaccharideendotoxin from Escherichia coli ( Difco ). The method described by Kakiuchi and Hall" was used to measure the effect of added endotoxin on the concentrations of cyclic 3'5' AMP. Metaoxedrine alone did not cause any contraction, but when the muscle was pre-treated with the beta-blocker sota!ol we obtained a slight contraction which increased with increasing doses of metaoxedrine. After the addition, of bacterial endotoxin, the constricting effect of-rnetaoxedrtne increased two to ten times in muscles from nonobstructive
patients. In preparation from a patient with chronic bronchitis, endotoxin potentiated the alpha-mediated constriction more than 1000 times (Fig 1) . The endotoxin also increased sensitivity to histamine. The addition of endotoxin to the muscle preparations from three patients with normal bronchi decreased the amount of cyclic AMP more than 60 percent. Endotoxin lowered the concentrations of CAMP in the bronchial muscles, which can explain the potentiation of the alpha-receptor mediated contraction. The lowering can be due to either a reduction of the adenyl cyclase activity (decreased synthesis of CAMP) or to an activation of phosphodiesterase (increased breakdown of CAMP); both would potentiate the muscle-contracting effect." If endotoxin can potentiate the a-mediated bronchial contractility also in vivo, this might explain the increased tendency to airway constriction seen in connection with bronchial infections. REFERENCES
Szentivanyi A: The beta-adrenergic theory of the atopic abnonnality in bronchial asthma. J Allerg 42:203-232, 1968. 2 Simonsson BG, Andersson R, Bergh NP, et al: In vivo and in vitro studies of pharmacological effects on different receptors regulating bronchial tone in man . In : Bronchitis III, Proceedings of the Third International Symposium on Bronchitis at Groningen, 1969. Assen, The Netherlands, Royal Van Gorcum, 1970, 334-342 3 Lundholm L, Mohme-Lundholm E: Length of inactivated contractile elements, length-tension diagram. Active state and tone of vascular smooth muscle. Acta Physiol Scand 68:347-359, 1966 4 Kakiuchi S, Rail TW: The influence of chemical agents on the accumulation of adenosine 3'5'-phosphate in slices of rabbit cerebellum. Mol Phannacol 4 :367-378, 1968 5 Andersson R, Nilsson K: Relaxation in intestinal smooth muscle; role of cyclic AMP and calcium . Nature (in press)
CHEST, VOL. 63, NO.4, APRIL 1973 SUPPLEMENT
15TH ANNUAL ASPEN CONFERENCE
sone; 2) systemic corticosteroid administration restores the responsiveness of adenyl cyclase to betaadrenergic stimulation; 3) systemic corticosteroids inhibit the increased ATPase activity, possibly with a concomitant decrease in transport, contractile and secretory processes; and 4) corticosteroids at high levels may inhibit cyclic AMP phosphodiesterase activity. The data also suggest that the therapeutic use of alpha-adrenergic blocking agents such as phentolamine, shown to be effective against asthma in Europe," should be studied clinically.
4
5
6
7 REFERENCES
1 Szentivanyi A: The beta-adrenergic theory of the atopic abnormality in bronchial asthma. J Allergy 42:203, 1968 2 Townley RG, Hanrath T, Guirgis HM : The inhibitory effect of hydrocortisone on the alpha adrenergic responses of human and guinea pig isolated respiratory smooth muscle. J Allergy Clin Immunol 49 :88, 1972 3 Coffey RG, Hadden JW, Hadden EM, et al l Stimulation of
8
9
ATPase by norepinephrine : An alpha-adrenergic receptor mechanism. Fed Proc 30:497, 1971 Bourne HR, Melmon KL: Adenyl cyclase in human leukocytes : eVidence for activation by separate beta-adrenergic and prostaglandin receptors. J Pharm Exper Therap 178: 1, 1971 Logsdon PJ, Middleton E Jr, Coffey RG: Stimulation of leukocyte adenyl cyclase by hydrocortisone and isoproterenol in asthmatic and non-asthmatic subjects. J Allergy Clin Immunol (in press) Logsdon PJ, Camright DV, Middleton E jr, et al : Effects of theophylline and aminophylline on stimulation of leukocyte adenyl cyclase in normal and asthmatic subjects. Biochem Biophys Res Commun (in press) Hadden JW, Hadden EM, Wilson EE, et al l Direct action of insulin on plasma membrane ATPase in human leukocytes. Nature, 235: 174, 1972 Orange RP, Kaliner MA, LaRaia PJ, et al : Immunologic release of histamine and slow reacting substance (Jf anaphylaxis from human lung . Influence of cellular levels of cyclic AMP Fed Proc 30:1725,1971 Marcelle R, Bottin R, Juchmes J, et al : Reactions bronchomotrica des receptors beta-adrenergiques. Acta Allergol 23:11,1968
In vivo and in vitro Studies on Alpha-receptors in Human Airways; Potentiation with Bacterial Endotoxins· Bo G. Simonsson, M.D.; N. Svedmyr, M.D.; B. -E. Skoogh, M.D.;
R. Anderson, M.D.; and N. P. Bergh, M.D.
The increased responsiveness of the airway caliber-regulating system seen in asthmatics may be due to increased sensitivity of the afferent nerve endings, ie the irritant receptors, or of the smooth bronchial muscle receptors. SzentivanyP has proposed a theory of functional beta-receptor blockade as a cause of airway obstruction in disease. A possible alpha-receptor stimulation would then cause bronchoconstriction. Studies in animals have shown the presence of alpha-receptors in the airway muscles; in Croningen in 1969 we gave a preliminary report on the first simultaneous evidence in vivo and in vitro of the presence of bronchoconstricting alphareceptors in human bronchial muscle." We have studied the effect in vivo of cholinergic blockade, adrenergic beta-blockade and alpha stimulation in 15 patients, most with airway ob°From the Department of Pharmacology, the Thoracic Surgery Clinic and the Lung Clinic, Benstromska Siukhuset, University of Coteborg, Sweden. o °Present address : University Lung Clinic ; Lasarettet, S-221 85 Lund, Sweden.
struction. In vitro studies on human bronchial muscles confirm the findings and show potentiation of alpha-receptor bronchoconstriction after treatment with endotoxin. We measured airway conductance (Caw) at different lung volumes in a volume-displacement body plethysmograph and computed conductance lines from which a computer compared Caw at specific subvolumes of the control total lung capacity (20, 55 and 80 percent VC) . We measured conductance during five-minute periods in the control state, ten minutes after IV injection of atropine (cholinergic blockade), 5-15 minutes after IV propranalol (,B-blockade), and 5-10 minutes after inhalation of metaoxedrine, a specific a-receptor stimulator.
Results in vivo Atropine caused bronchodilation in most subjects. Propranolol induced varying responses, the mean Caw was unchanged compared to the study post-
CHEST, VOL. 63, NO.4, APRIL 1973 SUPPLEMENT
45
15TH ANNUAL ASPEN CONFERENCE Q.Q2
i o,) ~f ~
0
•
0
1
.~a PHE
•
I
3min
5''d 1
1.7.ifJ PHE
EHDOTOX.
6 ' Jrnin
J
;
Q.02~ ~O.l ~o
o
3
3
I min 0 • • min 1. The effect of metaoxedrine (PHE) in vitro before and after addition of bacterial endotoxins on a previously not ,a-blocked human bronchial muscle . Below: Increased sensitivity for histamine in the same preparations.
FIGURE
atropine. After blockade of the beta-receptors and the vagal reflex, metaoxedrine induced a decrease of Caw in all but two subjects. The mean Gaw between the study after propranolol and the study after metaoxedrine was 0.17 (I/s)cm H20 (P
Results in vitro The in vitro studies were performed on specimens of segmental bronchi of 3-5 mm diameter obtained from patients undergoing operation for lung tumor. Preparations with a breadth of 5-10 mm and length of about 15 mm were then mounted in plastic holders according to Lundholm and Mohme-Lundholm." Tension changes of the circular muscle layer were measured with a Statham transducer on a Crass polygraph. The muscle was suspended in a buffer solution at 37° C and the various drugs were tested after adequate flushing of the preparations. The alpha-mediated bronchoconstriction was studied after treatment with a lipo-polysaccharideendotoxin from Escherichia coli ( Difco ). The method described by Kakiuchi and Hall" was used to measure the effect of added endotoxin on the concentrations of cyclic 3'5' AMP. Metaoxedrine alone did not cause any contraction, but when the muscle was pre-treated with the beta-blocker sota!ol we obtained a slight contraction which increased with increasing doses of metaoxedrine. After the addition, of bacterial endotoxin, the constricting effect of-rnetaoxedrtne increased two to ten times in muscles from nonobstructive
patients. In preparation from a patient with chronic bronchitis, endotoxin potentiated the alpha-mediated constriction more than 1000 times (Fig 1) . The endotoxin also increased sensitivity to histamine. The addition of endotoxin to the muscle preparations from three patients with normal bronchi decreased the amount of cyclic AMP more than 60 percent. Endotoxin lowered the concentrations of CAMP in the bronchial muscles, which can explain the potentiation of the alpha-receptor mediated contraction. The lowering can be due to either a reduction of the adenyl cyclase activity (decreased synthesis of CAMP) or to an activation of phosphodiesterase (increased breakdown of CAMP); both would potentiate the muscle-contracting effect." If endotoxin can potentiate the a-mediated bronchial contractility also in vivo, this might explain the increased tendency to airway constriction seen in connection with bronchial infections. REFERENCES
Szentivanyi A: The beta-adrenergic theory of the atopic abnonnality in bronchial asthma. J Allerg 42:203-232, 1968. 2 Simonsson BG, Andersson R, Bergh NP, et al: In vivo and in vitro studies of pharmacological effects on different receptors regulating bronchial tone in man . In : Bronchitis III, Proceedings of the Third International Symposium on Bronchitis at Groningen, 1969. Assen, The Netherlands, Royal Van Gorcum, 1970, 334-342 3 Lundholm L, Mohme-Lundholm E: Length of inactivated contractile elements, length-tension diagram. Active state and tone of vascular smooth muscle. Acta Physiol Scand 68:347-359, 1966 4 Kakiuchi S, Rail TW: The influence of chemical agents on the accumulation of adenosine 3'5'-phosphate in slices of rabbit cerebellum. Mol Phannacol 4 :367-378, 1968 5 Andersson R, Nilsson K: Relaxation in intestinal smooth muscle; role of cyclic AMP and calcium . Nature (in press)
CHEST, VOL. 63, NO.4, APRIL 1973 SUPPLEMENT