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Response of Avian Intrapulmonary Smooth Muscle to Changes in Carbon Dioxide Concentration1,2
Response of Avian Intrapulmonary Smooth Muscle to Changes in Carbon Dioxide Concentration!.2 G. M. BARNAS and F. B. MATHER Department of Animal Science, University of Nebraska, Lincoln, Nebraska 68583 and M. R. FEDDE Department of Anatomy and Physiology, Kansas State University, Manhattan, Kansas 66506
ABSTRACT How smooth muscle, at the openings of paleopulmonic parabronchi into the mediodorsal secondary bronchi, responds to changes in intrapulmonary C 0 2 concentrations was studied in spontaneously breathing and in paralyzed, unidirectionally ventilated ducks and geese. In the study, photographs of the parabronchi (taken through a small hole in the intercostal muscles and mediodorsal secondary bronchial wall) were analyzed. Also measured were changes in resistance to airflow through part of the lung in response to various CO, concentrations in the ventilatory gas. There were no consistent changes in the cross-sectional area of these parabronchi to changes in concentrations of C 0 2 entering the lung; however, the parabronchial smooth muscle frequently contracted spontaneously. Contractions could also be readily induced by stimulating the smooth muscle mechanically or the vagus nerve electrically. None of the various bronchodilator drugs tested successfully inhibited spontaneous or mechanically induced contractions, but atropine sulfate blocked the contraction caused by vagal stimulation. Although the smooth muscle in this part of the lung can contract rapidly, it probably does not do so in response to changes in intrapulmonary CO, concentration during eupneic breathing.
INTRODUCTION T h e avian lung contains an a b u n d a n c e of s m o o t h muscle (King and Cowie, 1 9 6 9 ; King a n d Molony, 1 9 7 1 ) . T h e large spiral muscle bands and t h e small oblique bundles associated with the secondary b r o n c h i and p a r a b r o n c h i m a y alter t h e d i a m e t e r of t h e lumina of t h e bronchi, t h e r e b y regulating t h e gas flow t h r o u g h t h e m . Such regulation m a y b e importa n t in shunting gas t h r o u g h nongas exchange regions of t h e lung during hyperventilation induced b y h e a t stress or in preventing imbalances in ventilation/perfusion of local regions of t h e lung. In chickens a n d ducks, carbon d i o x i d e changes in i n t r a p u l m o n a r y gas alter resistance t o gas flow t h r o u g h t h e lungs (Ray and Fedde, 1 9 6 9 ; Molony et al, 1 9 7 6 ) . F u r t h e r m o r e , Leit-
' Supported in part by a grant-in-aid from the American Heart Association, Kansas Affiliate, Inc. 2 Published as paper No. 5469, Journal Series, Nebraska Agricultural Experiment Station and Contribution No. 78-109-j, Department of Anatomy and Physiology, KAES, Kansas State University, Manhattan, Kansas 66506. 1978 Poultry Sci 57:1400-1407
ner a n d R o u m y ( 1 9 7 4 ) observed t h a t t h e m e d i o d o r s a l secondary b r o n c h i a n d parabronchi of ducks c o n t r a c t strongly during hypercapnia. T h u s , t h e changes in CO2 c o n c e n t r a t i o n in t h e i n t r a p u l m o n a r y gas during eupneic b r e a t h i n g m a y b e i m p o r t a n t in regulating t h e degree of c o n t r a c t i o n of intrapulmonary s m o o t h muscle. We e x a m i n e d and here report o n t h e response of t h e s m o o t h muscle in a particular lung region of ducks and geese (the openings of t h e p a l e o p u l m o n i c parabronchi i n t o t h e mediodorsal secondary bronchi) t o changes in C 0 2 c o n c e n t r a t i o n ; our aim was t o provide additional insight into t h e role of CO2 in controlling t h e avian respiratory system.
METHODS We studied 18 adult, domestic geese (mixed forms of Anser anser) ranging in b o d y weight from 3.1 t o 7.4 kg and 28 a d u l t w h i t e Pekin or R o u e n ducks (mixed forms of Anas platyrhynochos), 1.9 t o 2.9 kg. Animal Preparation. Each bird was anesthetized by injecting sodium p e n t o b a r b i t a l (10— 2 0 mg/kg) i n t o a c a n n u l a t e d c u t a n e o u s ulnar
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(Received for publication December 13,1977)
INTRAPULMONARY SMOOTH MUSCLE AND COa
Springs, model 72) was inserted about 10 cm into the rectum. The controller varied the current to an infrared lamp to keep the rectal temperature constant at 41.5 C. A second thermistor, attached to a tele-thermometer (Yellow Springs, model 44), enabled us to observe body temperature directly. A pressure transducer (Statham, model PM15) was attached in the unidirectional ventilation system just prior to the clavicular cannula to indicate intrapulmonary pressure (Pip). A pressure change at that site indicated resistance in the bird's airways had changed. Ventilatory gas flow was measured with a pneumotachometer (Godart-Statham, type 17212) and F\co with an infrared C 0 2 analyzer (Beckman,2 model LB-2); both were recorded on the pen recorder. The concentration of C 0 2 in the gas leaving the exposure of the parabronchi ( F p c o ) was periodically monitored, to be sure trie smooth muscle in that region was in contact with the desired C 0 2 concentration. A thermistor positioned just prior to the clavicular cannula and attached to the tele-thermometer measured the temperature of the gas entering the bird. The exposed parabronchi were photographed through the lens of an operational dissecting microscope (Zeiss) set at highest magnification (40x ). The field of observation included one to six parabronchi.
Drugs Used. Drugs known to cause dilation or prevent constriction of smooth muscle were used in an attempt to block completely bronchial smooth muscle activity. They were either given as a single injection or continuously infused intravenously, depending on the drug and dosage. The drugs and the maximum dosages used are shown in Table 1. We also used combinations of these drugs: terbutaline and propranolol; diphenhydramine and epinephrine; verapamil and isoproternol; verapamil, diphenhydramine, isoproternol, terbutaline, and epinephrine; diphenhydramine, verapamil, and aminophylline; papaverine and aminophylline; verapamil, diphenhydramine, aminophylline FIG. 1. Schema of the experimental arrangement and papaverine; and papaverine and atrophine for studying the response of parabronchial smooth muscle to changes in C0 2 concentrations in geese and sulfate. Not all drugs were used at their maximum dosages when used in combinations. ducks. M(j, PD, and Mv indicate mediodorsal secondary bronchi, parabronchi, and medioventral secondary Protocol. Each goose was exposed to alternabronchi, respectively; PArt> arterial blood pressure; ting periods of 0% and 3, 5, 8, 8.5, 9, 9.5, or V, flow of gas; T)j, rectal temperature; Tgas, tempera- 10% F i , during constant gas flow through co ture of gas; Fico , FEco >• an<^ FPCO » fractional concentration of inspired, expired, and parabronchial the lung. Parabronchi were photographed during 4 to 12 successive periods, after at least one C0 2 , respectively.
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vein. Throughout the experiment we made supplemental injections as needed, and all drugs administered subsequently were through this cannula. The clavicular and left caudal thoracic air sacs were cannulated, the trachea was occluded, and each bird was unidirectionally ventilated (Fedde et al., 1974) through the clavicular cannula with a heated (41.5 C), humidified mixture of oxygen (30%), carbon dioxide (various amounts), and nitrogen (various amounts) at a flow of 4 L/min (Fig. 1). By an arrangement of solenoid valves, which could introduce a compensatory volume of nitrogen (Fedde et al., 1974), the concentration of CO2 in the ventilatory gas ( F i c o ) could be switched instantly from a given level to zero and returned to the original level without changing flow. Flaxedil (.5—1.0 mg/kg) was injected intravenously to inhibit skeletal muscle activity and was supplemented as needed. After making a mid-thoracic skin incision and retracting the left scapula, we exposed a small portion (about 1 cm 2 ) of the first, second, or third mediodorsal secondary bronchus to observe directly the paleopulmonic parabronchi. We then sealed the exposure with damp cotton or bonewax if subsequent exposures were to be made. Recordings. Blood pressure was sensed through a brachial arterial cannula attached to a pressure transducer (Statham, model P23Gb) and recorded by a multichannel pen recorder (Brush, model 481). A thermistor leading to a proportional temperature controller (Yellow
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TABLE 1.—Drugs and maximum dosages used in an attempt to block parabronchial smooth muscle contraction Dosage
Isoproternol (Winthrop) Propranolol (Ayerst) Epinephrine (Gotham) Terbutaline (Astra) Atropine sulfate (Elkins-Sinn) Diphenhydramine (Parke-Davis) Aminophylline (Searle) Aminopropazine (Gen-Sal) Papaverine (Eli Lilly) Verapamil (Knoll) NaNOj
127jug/kg .14 mg/kg 3 jug/kg/min .51 mg/kg 1.2 mg/kg 3 0 mg/kg 100 mg/kg 3 mg/kg 43 mg/kg 15 mg/kg 100 mg/kg
minute from the beginning of each period. This procedure was usually repeated using several levels of F i r o , and usually at more than one site of parabronchial exposure. The ducks were subjected to a randomized protocol of Fi„ treatments, followed by several alternating 2 periods of 0% and 7% F j C Q . Pictures were taken after at least one minute from the beginning of each period. In some birds, the smooth muscle surrounding the parabronchial entrance was gently stimulated with a bristle to test its sensitivity to mechanical stimuli. Changes in Pip, which indicated possible changes in airway resistance caused by bronchial smooth muscle movements, were also examined. Drugs were then administered in an attempt to inhibit completely smooth muscle activity. We examined the response of the bronchial smooth muscle to vagal stimulation in two geese. The peripheral end of the cut left cervical vagus was electrically stimulated with a voltage of 5 or 15 volts, a stimulus frequency of 60 Hz, and a stimulus duration of 1.2 msec. Exposed parabronchi were photographed at various times before, during, and after stimulation. Exposed parabronchi were also photographed at the peak of inspiration or expiration during spontaneous breathing before artificial ventilation and injection of Flaxedil to determine if the parabronchial lumen changed its diameter during a respiratory cycle. Data Analysis. The photographic negatives were enlarged and used to outline individual parabronchial lumina on paper. We measured the areas of the lumina with a planimeter; and, using a calibration factor calculated from a pic-
F
ico2 RESULTS
Smooth Muscle Sensitivity to Various 02 Concentrations. We observed no consistent changes in the size of parabronchial lumina in response to changes in F I f , 0 in any of the 18 geese studied (Fig. 2). Phonographs taken of parabronchi in 10 of the geese were analyzed to determine if changes in C 0 2 concentrations resulted in changes in areas too small to be detected visually. A variety of changes in Fic„ were analyzed; a particular treatment was repeated in some cases but was used only once in others. The cross-sectional areas of only three of 48 parabronchi studied showed significant constriction during 10%, as compared with 0%FIco The relative change in cross-sectional area at each F I c o , compared with that at 0% F I c o , was plotted for 52 parabronchi. The slope 6f the resulting line was 0 (Fig. 3A). The wide variance in the area of an individual parabronchial lumen resulted from spontaneous contraction and relaxation of its surrounding smooth muscle. In addition to the spontaneous contractions (discussed in the next section), occasionally particular areas of parabronchial smooth muscle were observed to contract or relax slightly, then remain in that condition throughout the remaining observations. In almost all geese, when alternating periods of 0% and some higher F i c o were introduced, pulse pressure and blood pressure regularly decreased during the 0% periods after about a 10sec delay. During these alternating periods, Pip occasionally would show a consistent pattern of slight increases (about .5 mm H 2 0 ) in the 0% periods. In some ducks, the parabronchial smooth muscle appeared to be contracting with increased F i c o , but relaxation was also occasionally observed. Only four of 48 parabronchi constricted significantly, relative to their cross-
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Drug
ture of a millimeter scale taken at identical magnification, we could determine the crosssectional area of each parabronchus. To test the effects of changes in C 0 2 concentration in the ventilating gas on crosssectional area of each parabronchus, we used analysis of variance. If a significant difference among treatments was found (P<.05), the Duncan's Multiple Range Test was used to determine in which F[ treatment the crosssectional area differed from that found at 0%
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FIG. 2. Photographs of parabronchi during alternating 1-min periods of 7% and 0% F i ^ g • Pictures were taken at the end of three consecutive intervals of F l c o • indicated by the arrows. Bar in each photograph indicates .5 mm.
sectional area at 0% F k:o, with increased C 0 2 ; two others dilated significantly. The plot of the relative changes in the cross-sectional areas of 52 parabronchi at a given F\ca level, compared with 0%, gave a slope of —1?8 (not significantly different from 0), and an insignificant correlation of .1 (Fig. 3B). Spontaneous Contractions. As observed directly, the smooth muscle around the openings of all parabronchi in view occasionally would quickly contract then gradually relax. These contractions occurred spontaneously during any F i c _ treatment; they sometimes varied in intensify. At every observation of a spontaneous contraction, a sharp rise, followed by a gradual fall,
in the Pip was recorded (Fig. 4). Thus, the parabronchi appeared to contract spontaneously throughout the lung, resulting in a generalized increase in airway resistance. In all birds, these spontaneous contractions of parabronchial smooth muscle appeared and ceased for no discernable reason. In a few birds, an apparent regular pattern of contractions was exhibited. In one bird, contractions occurred about every nine minutes for a period of one hour. Infrequently, a contraction appeared to be potentiated by changes in F I c o ; that is, during alternating periods of C 0 2 , a contraction would regularly be observed at the beginning of one of the treatments, whether at 0% or some other level.
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FIG. 3. Changes in cross-sectional area of parabronchi, compared with their area at 0% F J Q Q function of the concentration of C0 2 entering the lung.
(A/A 0 ), as a
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Pip
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No spontaneous contractions were observed in the two geese whose left vagi were sectioned. Response to Mechanical Stimulation. Slightly touching the wall of the parabronchial lumen with a bristle caused the smooth muscle surrounding its opening to contract strongly (Fig. 5). After about one minute, the muscle would begin to relax. Repeated stimulation appeared to diminish the intensity of the response and prolong the time before relaxation. This response, which occurred in every parabronchus tested, was independent of changes in F i c _ and was not abolished by vagotomy. No changes in Pip were evident during mechanical stimulation. Effectiveness of Drugs in Blocking Smooth Muscle Activity. No single drug diminished the response to mechanical stimulation or prevent-
ed spontaneous contractions of parabronchial smooth muscle. Increases in heart rate were induced by isoproternol, epinephrine, aminophylline, and papaverine. Diastolic blood pressure was lowered by aminophylline, verapamil, and papaverine; it was greatly increased by epinephrine. Diphenhydramine caused blood pressure to decrease immediately, then return to normal within one minute. Combinations of 24 mg/kg body weight diphenhydramine, 9.5 mg/kg verapamil, and 90 mg/kg aminophylline; 25.7 mg/kg papaverine and 107 mg/kg aminophylline; or 16 mg/kg verapamil, 36 mg/kg diphenhydramine, 100 mg/ kg aminophylline, and 18 mg/kg papaverine rendered some regions of parabronchial smooth muscle unreactive to mechanical stimulation, but in each bird parabronchi in some areas re-
FIG. 5. Response of parabronchi in a goose to mechanical stimulation. A, before stimulation; B, the parabronchus to the far right of the picture was stimulated with a bristle 1 min after A; C, the parabronchus in the center was stimulated 1 min later. Bar in each photograph indicates .5 mm.
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FIG. 4. Changes in the cross-sectional area of parachonchi and in Pip in a goose during a spontaneous contraction of parabronchi smooth muscle. A, before contraction; B, at the peak of contraction; C, 165 sec following contraction. Bar in each photograph indicates .5 mm.
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FIG. 6. Responses of parabronchi in a goose to electrical stimulation of the vagus nerve before (A, B, C) and after (D, E, F) injecting 3.5 mg atropine sulfate. A, 20 sec before stimulation; B, after 3 sec of 5-volt stimulation; C, after 50 sec of continual 5-volt stimulation; D, 30 sec before stimulation; E, after 3 sec of 5-volt stimulation; F, during 15-volt stimulation, 2 hr after atropine sulfate injection. Bar in each photograph indicates .5 mm.
sponded. Pip patterns indicated that spontaneous contractions were absent or greatly reduced in magnitude in birds subjected to any of these drug combinations, but because the contractions appeared irregularly before drugs were given, we cannot say conclusively that any drug treatment inhibited them. Response to Peripheral Vagal Stimulation and the Effect of Atropine. When the vagus nerve was stimulated in two geese, all parabronchi exposed in three different sites in each goose reacted similarly. Stimulating the peripheral end of the cut left vagus reduced the area of the parabronchial lumina as the smooth muscle surrounding each contracted markedly (Fig. 6A, B). The smooth muscle began to relax after a few seconds, even when stimulation was continued (Fig. 6C). The procedure was repeated during 0%, 5%, and 10% FT with identical results. The heart CO, would momentarily stop, then recover, during the electrical stimulation. We observed an increase in Pip, similar in magnitude to the in-
creases found during a spontaneous contraction, during vagal stimulation. A minimum of 1 mg of atropine sulfate completely inhibited the parabronchial response to vagal stimulation (Fig. 6D, E), although with mechanical stimulation, the parabronchial smooth muscle contracted normally. About one hour after injecting atropine sulfate, we occasionally observed slight movement of the smooth muscle during stimulation (Fig. 6F). The vagus was still capable of conducting impulses at that time, as indicated by small changes in heart rate during the periods of stimulation. No changes in Pip were noted during these post-drug stimulations. Response of Smooth Muscle to Changes in Unidirectional Gas Flow. In three geese, gas flow was decreased from 4 liters/min to 2 liters/ min during 0% F i c o . Measurements from photographs showed no consistent changes in the cross-sectional areas of the 10 parabronchi measured. Changes in Parabronchial Lumen during
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Spontaneous Breathing. No consistent change related to inspiration or expiration was observed in the cross-sectional area of any of the 18 parabronchi analyzed in five geese. DISCUSSION
Viability of the Exposed Smooth Muscle. The occasional spontaneous contractions, responses to mechanical stimulation, contractions during vagal stimulation, and the wide variance in an individual parabronchial lumen's crosssectional area (Fig. 3) indicate that the exposed parabronchial smooth muscle remained viable throughout the experiment. Hence, lack of response of the smooth muscle to changes in C0 2 concentration was not due to lung deterioration or inability of the muscle to contract. Parabronchial Smooth Muscle Contraction and FIQO2 • Our data indicate that smooth muscle contractibility in the parabronchi in geese or ducks does not change consistently when intrapulmonary CO2 concentrations are changed over a wide range. Mammalian studies have shown that CO2 has a variable effect on airway-muscle tone, influencing it by more than one mechanism. Nadel and Widdicombe (1962) reported that airways constricted in intact dogs and cats ventilated with 8% CO2, a response abolished by vagotomy. In contrast, several workers (Nisell, 1950; Severinghaus et al., 1961; Astin et al., 1973; Duckies et al, 1974) have shown that C0 2 has a direct bronchodilatory effect on air-
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Accuracy of Measurements. In measuring parabronchial lumina, there was a small random error of ± .01 mm 2 (due to difficulty in outlining a parabronchial lumen on paper and to limitations of the planimeter). To minimize this error, we were careful to outline the same corresponding layer of smooth muscle around each lumen and to repeat each planimeter measurement then taking the average. We doubt, however, that changes in area too small to be detected were of physiological significance, in that responses to changes in C 0 2 concentration were inconsistent; many parabronchi constricted and dilated in response to repetitive identical F I C Q treatments. Activity of Bronchial Smooth Muscle in Parts of the Lung Not Observed Directly. The similar response of the randomly selected parachronchi between the second and fifth ribs suggests that all parabronchial smooth muscle reacts to F I C Q changes similarly. There is no anatomical evidence for specialized parabronchial areas that may act differently (King and Cowie, 1969). Spontaneous and electrically induced contractions always produced increases in Pip, indicating generalized contraction of the smooth muscle throughout the lung. Thus, similar pressure increases would have been expected if there had been a generalized contraction of parabronchial smooth muscle with changes in FlCO • The only pressure changes we observed were slight increases in Pip when F i c o was 0%. Though these pressure changes did not correlate with a measured reduction in the parabronchial lumina, they indicated that muscular contraction had occurred at some site in the lung. Ray and Fedde (1969) recorded large decreases in tracheal pressure as F i c o increased in unidirectionally ventilated chickens. Molony et al. (1976) reported that lowering F i c o from 5% in unidirectionally ventilated ducks markedly increased lung resistance (the value at 0% F i c o w a s more than twice that at 5%). They presented evidence that constriction of the openings of the medioventral secondary bronchi into the primary bronchus are mainly responsible for the change, while "the resist-
ance offered by the parabronchi appeared to be smaller and less dependent on CO2." In our study, gas was directed into the clavicular air sac, which arises by large ducts from the first, second, and third medioventral secondary bronchi; most of the gas should have thereby gone directly to the paleopulmonic parabronchi. The high resistance site at the entrance of the medioventral bronchi into the intrapulmonary primary bronchus was largely by-passed, so our resistance measurements probably were more sensitive to constriction in those parabronchi than if gas had been directed into the lung through the trachea. However, the slight increase in Pip during 0% F i C o might have resulted from constriction of the orifices of the medioventral secondary bronchi, thereby increasing the resistance to flow in one pathway. Gaseous Environment of Exposed Smooth Muscle. Measurements of F c o 2 at the site of parabronchial observation indicated that at least the outermost layers of smooth muscle surrounding the parabronchi were subjected to the desired concentrations of CO2. Because these outer layers were used to outline the parabronchial lumen from the photographs, they were the most influential in the measurements analyzed.
INTRAPULMONARY SMOOTH MUSCLE AND C0 2 w a y s previously c o n s t r i c t e d b y a n y of several different stimuli, including drugs or p u l m o n a r y artery occlusion. Ingram ( 1 9 7 5 ) , w h o separated t h e effects of changes in arterial CO2 and airway CO 2 in t h e dog, f o u n d t h a t decreases from n o r m a l airway CO2 increased lung resistance and t h a t , conversely, increases in arterial CO2 were directly related t o increases in resistance which were vagally m e d i a t e d .
Both King and Cowie ( 1 9 6 9 ) and Lewis ( 1 9 2 4 ) observed r e p e a t e d r h y t h m i c contractions of avian bronchial s m o o t h muscle in vivo and in vitro after t h e y applied e x c i t a t o r y drugs; t h e contractions were arrested b y a t r o p i n e . T h e s p o n t a n e o u s c o n t r a c t i o n s we observed occurred irregularly. Our results indicated t h a t t h e contractions could b e abolished b y v a g o t o m y b u t could n o t b e totally inhibited by atropine. T h u s , it is difficult t o ascertain w h e t h e r t h e s p o n t a n e o u s c o n t r a c t i o n s were neurally induced or resulted from s o m e intrinsic p r o p e r t y of t h e s m o o t h muscle.
ACKNOWLEDGMENTS We t h a n k Wade K u h l m a n n , Dean Klentz, Dalyn Wilson, and David A d a m s for their technical assistance and helpful advice.
REFERENCES Astin, T. W., G. R. Barer, J. W. Shaw, and P. M. Warren, 1973. The action of carbon dioxide on constricted airways. J. Physiol. (London) 2 3 5 : 6 0 7 623. Duckies, S. P., M. D. Rayner, and J. A. Nadel, 1974. Effects of C0 2 and pH on drug-induced contractions of airway smooth muscle. J. Pharm. Exp. Ther. 190:472-481. Fedde, M. R., R. N. Gatz, H. Slama, and P. Scheid, 1974a. Intrapulmonary C 0 2 receptors in the duck: I. Stimulus specificity. Respir. Physiol. 22:99— 114. Ingram, R. H., 1975. Effects of airway versus arterial C0 2 changes on lung mechanics in dogs. J. Appl. Physiol. 38:603-607. King, A. S., and A. F. Cowie, 1969. The functional anatomy of the bronchial muscle of the bird. J. Anat. 105:323-336. King, A. S., and V. Molony, 1971. The anatomy of respiration. Page 93—169 in Physiology and biochemistry of the domestic fowl. D. J. Bell and B. M. Freeman, ed. Academic Press, New York. Leitner, M.-M., and M. Roumy, 1974. Vagal afferent activities related to the respiratory cycle in the duck: Sensitivity to mechanical, chemical and electrical stimuli. Respir. Physiol. 22:41—56. Lewis, M. R., 1924. Spontaneous rhythmical contraction of muscles of the bronchial tubes and air sacs of the chick embryo. Amer. J. Physiol. 68:385— 388. Molony, V., W. Graf, and P. Scheid, 1976. Effects of C0 2 on pulmonary air flow resistance in the duck. Respir. Physiol. 26:333-349. Nadel, J. A., and J. G. Widdicombe, 1962. Effect of changes in blood gas tensions and carotid sinus pressure on tracheal volume and total lung resistance to flow. J. Physiol. (London) 163:13—33. Nisell, O. I., 1950. The action of oxygen and carbon dioxide in the bronchioles and vessels of the isolated perfused lung. Acta Physiol. Scand. 21:5—62. Ray, P. J., and M. R. Fedde, 1969. Responses to alterations in respiratory P o 2 and PC0 2 in the chicken. Respir. Physiol. 6:135-143. Severinghaus, J. W., E. W. Swenson, T. N. Finley, M. T. Lategola, and J. Williams, 1961. Unilateral hypoventilation produced in dogs by occluding one pulmonary artery. J. Appl. Physiol. 16:53—60.
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In our s t u d y , arterial CO2 was n o t monitored, so it was difficult t o estimate t h e i m m e d iate changes in t h e CO2 c o n c e n t r a t i o n of t h e blood a c c o m p a n y i n g changes in F i c o • However, it is reasonable t o assume t h a t t h e interaction of t h e t w o contrasting mechanisms — t h e direct action of F I C Q and t h e vagally mediated response t o arterial C 0 2 changes — c o n t r i b u t e d t o t h e wide variance in response of t h e parabronchial s m o o t h muscle (Fig. 3). Mechanism of the Smooth Muscle Responses. T h e response of t h e parabronchial s m o o t h muscle t o mechanical stimulation was n o t abolished b y v a g a t o m y ; t h a t suggests a localized intrinsic m e c h a n i s m , one n o t neurally mediated, was involved. Our observations of neurally induced contraction of t h e s m o o t h muscle during vagal stimulation agree with those of King and Cowie ( 1 9 6 9 ) and Leitner a n d R o u m y ( 1 9 7 4 ) . We further observed t h a t this response was blocked by a t r o p i n e , which c o m p e t e s with t h e excitat o r y t r a n s m i t t e r s u b s t a n c e , acetylcholine, for receptor sites a t p a r a s y m p a t h e t i c n e u r o m u s cular j u n c t i o n s .
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ABSTRACT How smooth muscle, at the openings of paleopulmonic parabronchi into the mediodorsal secondary bronchi, responds to changes in intrapulmonary C 0 2 concentrations was studied in spontaneously breathing and in paralyzed, unidirectionally ventilated ducks and geese. In the study, photographs of the parabronchi (taken through a small hole in the intercostal muscles and mediodorsal secondary bronchial wall) were analyzed. Also measured were changes in resistance to airflow through part of the lung in response to various CO, concentrations in the ventilatory gas. There were no consistent changes in the cross-sectional area of these parabronchi to changes in concentrations of C 0 2 entering the lung; however, the parabronchial smooth muscle frequently contracted spontaneously. Contractions could also be readily induced by stimulating the smooth muscle mechanically or the vagus nerve electrically. None of the various bronchodilator drugs tested successfully inhibited spontaneous or mechanically induced contractions, but atropine sulfate blocked the contraction caused by vagal stimulation. Although the smooth muscle in this part of the lung can contract rapidly, it probably does not do so in response to changes in intrapulmonary CO, concentration during eupneic breathing.
INTRODUCTION T h e avian lung contains an a b u n d a n c e of s m o o t h muscle (King and Cowie, 1 9 6 9 ; King a n d Molony, 1 9 7 1 ) . T h e large spiral muscle bands and t h e small oblique bundles associated with the secondary b r o n c h i and p a r a b r o n c h i m a y alter t h e d i a m e t e r of t h e lumina of t h e bronchi, t h e r e b y regulating t h e gas flow t h r o u g h t h e m . Such regulation m a y b e importa n t in shunting gas t h r o u g h nongas exchange regions of t h e lung during hyperventilation induced b y h e a t stress or in preventing imbalances in ventilation/perfusion of local regions of t h e lung. In chickens a n d ducks, carbon d i o x i d e changes in i n t r a p u l m o n a r y gas alter resistance t o gas flow t h r o u g h t h e lungs (Ray and Fedde, 1 9 6 9 ; Molony et al, 1 9 7 6 ) . F u r t h e r m o r e , Leit-
' Supported in part by a grant-in-aid from the American Heart Association, Kansas Affiliate, Inc. 2 Published as paper No. 5469, Journal Series, Nebraska Agricultural Experiment Station and Contribution No. 78-109-j, Department of Anatomy and Physiology, KAES, Kansas State University, Manhattan, Kansas 66506. 1978 Poultry Sci 57:1400-1407
ner a n d R o u m y ( 1 9 7 4 ) observed t h a t t h e m e d i o d o r s a l secondary b r o n c h i a n d parabronchi of ducks c o n t r a c t strongly during hypercapnia. T h u s , t h e changes in CO2 c o n c e n t r a t i o n in t h e i n t r a p u l m o n a r y gas during eupneic b r e a t h i n g m a y b e i m p o r t a n t in regulating t h e degree of c o n t r a c t i o n of intrapulmonary s m o o t h muscle. We e x a m i n e d and here report o n t h e response of t h e s m o o t h muscle in a particular lung region of ducks and geese (the openings of t h e p a l e o p u l m o n i c parabronchi i n t o t h e mediodorsal secondary bronchi) t o changes in C 0 2 c o n c e n t r a t i o n ; our aim was t o provide additional insight into t h e role of CO2 in controlling t h e avian respiratory system.
METHODS We studied 18 adult, domestic geese (mixed forms of Anser anser) ranging in b o d y weight from 3.1 t o 7.4 kg and 28 a d u l t w h i t e Pekin or R o u e n ducks (mixed forms of Anas platyrhynochos), 1.9 t o 2.9 kg. Animal Preparation. Each bird was anesthetized by injecting sodium p e n t o b a r b i t a l (10— 2 0 mg/kg) i n t o a c a n n u l a t e d c u t a n e o u s ulnar
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(Received for publication December 13,1977)
INTRAPULMONARY SMOOTH MUSCLE AND COa
Springs, model 72) was inserted about 10 cm into the rectum. The controller varied the current to an infrared lamp to keep the rectal temperature constant at 41.5 C. A second thermistor, attached to a tele-thermometer (Yellow Springs, model 44), enabled us to observe body temperature directly. A pressure transducer (Statham, model PM15) was attached in the unidirectional ventilation system just prior to the clavicular cannula to indicate intrapulmonary pressure (Pip). A pressure change at that site indicated resistance in the bird's airways had changed. Ventilatory gas flow was measured with a pneumotachometer (Godart-Statham, type 17212) and F\co with an infrared C 0 2 analyzer (Beckman,2 model LB-2); both were recorded on the pen recorder. The concentration of C 0 2 in the gas leaving the exposure of the parabronchi ( F p c o ) was periodically monitored, to be sure trie smooth muscle in that region was in contact with the desired C 0 2 concentration. A thermistor positioned just prior to the clavicular cannula and attached to the tele-thermometer measured the temperature of the gas entering the bird. The exposed parabronchi were photographed through the lens of an operational dissecting microscope (Zeiss) set at highest magnification (40x ). The field of observation included one to six parabronchi.
Drugs Used. Drugs known to cause dilation or prevent constriction of smooth muscle were used in an attempt to block completely bronchial smooth muscle activity. They were either given as a single injection or continuously infused intravenously, depending on the drug and dosage. The drugs and the maximum dosages used are shown in Table 1. We also used combinations of these drugs: terbutaline and propranolol; diphenhydramine and epinephrine; verapamil and isoproternol; verapamil, diphenhydramine, isoproternol, terbutaline, and epinephrine; diphenhydramine, verapamil, and aminophylline; papaverine and aminophylline; verapamil, diphenhydramine, aminophylline FIG. 1. Schema of the experimental arrangement and papaverine; and papaverine and atrophine for studying the response of parabronchial smooth muscle to changes in C0 2 concentrations in geese and sulfate. Not all drugs were used at their maximum dosages when used in combinations. ducks. M(j, PD, and Mv indicate mediodorsal secondary bronchi, parabronchi, and medioventral secondary Protocol. Each goose was exposed to alternabronchi, respectively; PArt> arterial blood pressure; ting periods of 0% and 3, 5, 8, 8.5, 9, 9.5, or V, flow of gas; T)j, rectal temperature; Tgas, tempera- 10% F i , during constant gas flow through co ture of gas; Fico , FEco >• an<^ FPCO » fractional concentration of inspired, expired, and parabronchial the lung. Parabronchi were photographed during 4 to 12 successive periods, after at least one C0 2 , respectively.
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vein. Throughout the experiment we made supplemental injections as needed, and all drugs administered subsequently were through this cannula. The clavicular and left caudal thoracic air sacs were cannulated, the trachea was occluded, and each bird was unidirectionally ventilated (Fedde et al., 1974) through the clavicular cannula with a heated (41.5 C), humidified mixture of oxygen (30%), carbon dioxide (various amounts), and nitrogen (various amounts) at a flow of 4 L/min (Fig. 1). By an arrangement of solenoid valves, which could introduce a compensatory volume of nitrogen (Fedde et al., 1974), the concentration of CO2 in the ventilatory gas ( F i c o ) could be switched instantly from a given level to zero and returned to the original level without changing flow. Flaxedil (.5—1.0 mg/kg) was injected intravenously to inhibit skeletal muscle activity and was supplemented as needed. After making a mid-thoracic skin incision and retracting the left scapula, we exposed a small portion (about 1 cm 2 ) of the first, second, or third mediodorsal secondary bronchus to observe directly the paleopulmonic parabronchi. We then sealed the exposure with damp cotton or bonewax if subsequent exposures were to be made. Recordings. Blood pressure was sensed through a brachial arterial cannula attached to a pressure transducer (Statham, model P23Gb) and recorded by a multichannel pen recorder (Brush, model 481). A thermistor leading to a proportional temperature controller (Yellow
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TABLE 1.—Drugs and maximum dosages used in an attempt to block parabronchial smooth muscle contraction Dosage
Isoproternol (Winthrop) Propranolol (Ayerst) Epinephrine (Gotham) Terbutaline (Astra) Atropine sulfate (Elkins-Sinn) Diphenhydramine (Parke-Davis) Aminophylline (Searle) Aminopropazine (Gen-Sal) Papaverine (Eli Lilly) Verapamil (Knoll) NaNOj
127jug/kg .14 mg/kg 3 jug/kg/min .51 mg/kg 1.2 mg/kg 3 0 mg/kg 100 mg/kg 3 mg/kg 43 mg/kg 15 mg/kg 100 mg/kg
minute from the beginning of each period. This procedure was usually repeated using several levels of F i r o , and usually at more than one site of parabronchial exposure. The ducks were subjected to a randomized protocol of Fi„ treatments, followed by several alternating 2 periods of 0% and 7% F j C Q . Pictures were taken after at least one minute from the beginning of each period. In some birds, the smooth muscle surrounding the parabronchial entrance was gently stimulated with a bristle to test its sensitivity to mechanical stimuli. Changes in Pip, which indicated possible changes in airway resistance caused by bronchial smooth muscle movements, were also examined. Drugs were then administered in an attempt to inhibit completely smooth muscle activity. We examined the response of the bronchial smooth muscle to vagal stimulation in two geese. The peripheral end of the cut left cervical vagus was electrically stimulated with a voltage of 5 or 15 volts, a stimulus frequency of 60 Hz, and a stimulus duration of 1.2 msec. Exposed parabronchi were photographed at various times before, during, and after stimulation. Exposed parabronchi were also photographed at the peak of inspiration or expiration during spontaneous breathing before artificial ventilation and injection of Flaxedil to determine if the parabronchial lumen changed its diameter during a respiratory cycle. Data Analysis. The photographic negatives were enlarged and used to outline individual parabronchial lumina on paper. We measured the areas of the lumina with a planimeter; and, using a calibration factor calculated from a pic-
F
ico2 RESULTS
Smooth Muscle Sensitivity to Various 02 Concentrations. We observed no consistent changes in the size of parabronchial lumina in response to changes in F I f , 0 in any of the 18 geese studied (Fig. 2). Phonographs taken of parabronchi in 10 of the geese were analyzed to determine if changes in C 0 2 concentrations resulted in changes in areas too small to be detected visually. A variety of changes in Fic„ were analyzed; a particular treatment was repeated in some cases but was used only once in others. The cross-sectional areas of only three of 48 parabronchi studied showed significant constriction during 10%, as compared with 0%FIco The relative change in cross-sectional area at each F I c o , compared with that at 0% F I c o , was plotted for 52 parabronchi. The slope 6f the resulting line was 0 (Fig. 3A). The wide variance in the area of an individual parabronchial lumen resulted from spontaneous contraction and relaxation of its surrounding smooth muscle. In addition to the spontaneous contractions (discussed in the next section), occasionally particular areas of parabronchial smooth muscle were observed to contract or relax slightly, then remain in that condition throughout the remaining observations. In almost all geese, when alternating periods of 0% and some higher F i c o were introduced, pulse pressure and blood pressure regularly decreased during the 0% periods after about a 10sec delay. During these alternating periods, Pip occasionally would show a consistent pattern of slight increases (about .5 mm H 2 0 ) in the 0% periods. In some ducks, the parabronchial smooth muscle appeared to be contracting with increased F i c o , but relaxation was also occasionally observed. Only four of 48 parabronchi constricted significantly, relative to their cross-
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Drug
ture of a millimeter scale taken at identical magnification, we could determine the crosssectional area of each parabronchus. To test the effects of changes in C 0 2 concentration in the ventilating gas on crosssectional area of each parabronchus, we used analysis of variance. If a significant difference among treatments was found (P<.05), the Duncan's Multiple Range Test was used to determine in which F[ treatment the crosssectional area differed from that found at 0%
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FIG. 2. Photographs of parabronchi during alternating 1-min periods of 7% and 0% F i ^ g • Pictures were taken at the end of three consecutive intervals of F l c o • indicated by the arrows. Bar in each photograph indicates .5 mm.
sectional area at 0% F k:o, with increased C 0 2 ; two others dilated significantly. The plot of the relative changes in the cross-sectional areas of 52 parabronchi at a given F\ca level, compared with 0%, gave a slope of —1?8 (not significantly different from 0), and an insignificant correlation of .1 (Fig. 3B). Spontaneous Contractions. As observed directly, the smooth muscle around the openings of all parabronchi in view occasionally would quickly contract then gradually relax. These contractions occurred spontaneously during any F i c _ treatment; they sometimes varied in intensify. At every observation of a spontaneous contraction, a sharp rise, followed by a gradual fall,
in the Pip was recorded (Fig. 4). Thus, the parabronchi appeared to contract spontaneously throughout the lung, resulting in a generalized increase in airway resistance. In all birds, these spontaneous contractions of parabronchial smooth muscle appeared and ceased for no discernable reason. In a few birds, an apparent regular pattern of contractions was exhibited. In one bird, contractions occurred about every nine minutes for a period of one hour. Infrequently, a contraction appeared to be potentiated by changes in F I c o ; that is, during alternating periods of C 0 2 , a contraction would regularly be observed at the beginning of one of the treatments, whether at 0% or some other level.
I A
:
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DUCKS 1
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: 1
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i
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5 F
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FIG. 3. Changes in cross-sectional area of parabronchi, compared with their area at 0% F J Q Q function of the concentration of C0 2 entering the lung.
(A/A 0 ), as a
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No spontaneous contractions were observed in the two geese whose left vagi were sectioned. Response to Mechanical Stimulation. Slightly touching the wall of the parabronchial lumen with a bristle caused the smooth muscle surrounding its opening to contract strongly (Fig. 5). After about one minute, the muscle would begin to relax. Repeated stimulation appeared to diminish the intensity of the response and prolong the time before relaxation. This response, which occurred in every parabronchus tested, was independent of changes in F i c _ and was not abolished by vagotomy. No changes in Pip were evident during mechanical stimulation. Effectiveness of Drugs in Blocking Smooth Muscle Activity. No single drug diminished the response to mechanical stimulation or prevent-
ed spontaneous contractions of parabronchial smooth muscle. Increases in heart rate were induced by isoproternol, epinephrine, aminophylline, and papaverine. Diastolic blood pressure was lowered by aminophylline, verapamil, and papaverine; it was greatly increased by epinephrine. Diphenhydramine caused blood pressure to decrease immediately, then return to normal within one minute. Combinations of 24 mg/kg body weight diphenhydramine, 9.5 mg/kg verapamil, and 90 mg/kg aminophylline; 25.7 mg/kg papaverine and 107 mg/kg aminophylline; or 16 mg/kg verapamil, 36 mg/kg diphenhydramine, 100 mg/ kg aminophylline, and 18 mg/kg papaverine rendered some regions of parabronchial smooth muscle unreactive to mechanical stimulation, but in each bird parabronchi in some areas re-
FIG. 5. Response of parabronchi in a goose to mechanical stimulation. A, before stimulation; B, the parabronchus to the far right of the picture was stimulated with a bristle 1 min after A; C, the parabronchus in the center was stimulated 1 min later. Bar in each photograph indicates .5 mm.
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FIG. 4. Changes in the cross-sectional area of parachonchi and in Pip in a goose during a spontaneous contraction of parabronchi smooth muscle. A, before contraction; B, at the peak of contraction; C, 165 sec following contraction. Bar in each photograph indicates .5 mm.
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FIG. 6. Responses of parabronchi in a goose to electrical stimulation of the vagus nerve before (A, B, C) and after (D, E, F) injecting 3.5 mg atropine sulfate. A, 20 sec before stimulation; B, after 3 sec of 5-volt stimulation; C, after 50 sec of continual 5-volt stimulation; D, 30 sec before stimulation; E, after 3 sec of 5-volt stimulation; F, during 15-volt stimulation, 2 hr after atropine sulfate injection. Bar in each photograph indicates .5 mm.
sponded. Pip patterns indicated that spontaneous contractions were absent or greatly reduced in magnitude in birds subjected to any of these drug combinations, but because the contractions appeared irregularly before drugs were given, we cannot say conclusively that any drug treatment inhibited them. Response to Peripheral Vagal Stimulation and the Effect of Atropine. When the vagus nerve was stimulated in two geese, all parabronchi exposed in three different sites in each goose reacted similarly. Stimulating the peripheral end of the cut left vagus reduced the area of the parabronchial lumina as the smooth muscle surrounding each contracted markedly (Fig. 6A, B). The smooth muscle began to relax after a few seconds, even when stimulation was continued (Fig. 6C). The procedure was repeated during 0%, 5%, and 10% FT with identical results. The heart CO, would momentarily stop, then recover, during the electrical stimulation. We observed an increase in Pip, similar in magnitude to the in-
creases found during a spontaneous contraction, during vagal stimulation. A minimum of 1 mg of atropine sulfate completely inhibited the parabronchial response to vagal stimulation (Fig. 6D, E), although with mechanical stimulation, the parabronchial smooth muscle contracted normally. About one hour after injecting atropine sulfate, we occasionally observed slight movement of the smooth muscle during stimulation (Fig. 6F). The vagus was still capable of conducting impulses at that time, as indicated by small changes in heart rate during the periods of stimulation. No changes in Pip were noted during these post-drug stimulations. Response of Smooth Muscle to Changes in Unidirectional Gas Flow. In three geese, gas flow was decreased from 4 liters/min to 2 liters/ min during 0% F i c o . Measurements from photographs showed no consistent changes in the cross-sectional areas of the 10 parabronchi measured. Changes in Parabronchial Lumen during
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Spontaneous Breathing. No consistent change related to inspiration or expiration was observed in the cross-sectional area of any of the 18 parabronchi analyzed in five geese. DISCUSSION
Viability of the Exposed Smooth Muscle. The occasional spontaneous contractions, responses to mechanical stimulation, contractions during vagal stimulation, and the wide variance in an individual parabronchial lumen's crosssectional area (Fig. 3) indicate that the exposed parabronchial smooth muscle remained viable throughout the experiment. Hence, lack of response of the smooth muscle to changes in C0 2 concentration was not due to lung deterioration or inability of the muscle to contract. Parabronchial Smooth Muscle Contraction and FIQO2 • Our data indicate that smooth muscle contractibility in the parabronchi in geese or ducks does not change consistently when intrapulmonary CO2 concentrations are changed over a wide range. Mammalian studies have shown that CO2 has a variable effect on airway-muscle tone, influencing it by more than one mechanism. Nadel and Widdicombe (1962) reported that airways constricted in intact dogs and cats ventilated with 8% CO2, a response abolished by vagotomy. In contrast, several workers (Nisell, 1950; Severinghaus et al., 1961; Astin et al., 1973; Duckies et al, 1974) have shown that C0 2 has a direct bronchodilatory effect on air-
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Accuracy of Measurements. In measuring parabronchial lumina, there was a small random error of ± .01 mm 2 (due to difficulty in outlining a parabronchial lumen on paper and to limitations of the planimeter). To minimize this error, we were careful to outline the same corresponding layer of smooth muscle around each lumen and to repeat each planimeter measurement then taking the average. We doubt, however, that changes in area too small to be detected were of physiological significance, in that responses to changes in C 0 2 concentration were inconsistent; many parabronchi constricted and dilated in response to repetitive identical F I C Q treatments. Activity of Bronchial Smooth Muscle in Parts of the Lung Not Observed Directly. The similar response of the randomly selected parachronchi between the second and fifth ribs suggests that all parabronchial smooth muscle reacts to F I C Q changes similarly. There is no anatomical evidence for specialized parabronchial areas that may act differently (King and Cowie, 1969). Spontaneous and electrically induced contractions always produced increases in Pip, indicating generalized contraction of the smooth muscle throughout the lung. Thus, similar pressure increases would have been expected if there had been a generalized contraction of parabronchial smooth muscle with changes in FlCO • The only pressure changes we observed were slight increases in Pip when F i c o was 0%. Though these pressure changes did not correlate with a measured reduction in the parabronchial lumina, they indicated that muscular contraction had occurred at some site in the lung. Ray and Fedde (1969) recorded large decreases in tracheal pressure as F i c o increased in unidirectionally ventilated chickens. Molony et al. (1976) reported that lowering F i c o from 5% in unidirectionally ventilated ducks markedly increased lung resistance (the value at 0% F i c o w a s more than twice that at 5%). They presented evidence that constriction of the openings of the medioventral secondary bronchi into the primary bronchus are mainly responsible for the change, while "the resist-
ance offered by the parabronchi appeared to be smaller and less dependent on CO2." In our study, gas was directed into the clavicular air sac, which arises by large ducts from the first, second, and third medioventral secondary bronchi; most of the gas should have thereby gone directly to the paleopulmonic parabronchi. The high resistance site at the entrance of the medioventral bronchi into the intrapulmonary primary bronchus was largely by-passed, so our resistance measurements probably were more sensitive to constriction in those parabronchi than if gas had been directed into the lung through the trachea. However, the slight increase in Pip during 0% F i C o might have resulted from constriction of the orifices of the medioventral secondary bronchi, thereby increasing the resistance to flow in one pathway. Gaseous Environment of Exposed Smooth Muscle. Measurements of F c o 2 at the site of parabronchial observation indicated that at least the outermost layers of smooth muscle surrounding the parabronchi were subjected to the desired concentrations of CO2. Because these outer layers were used to outline the parabronchial lumen from the photographs, they were the most influential in the measurements analyzed.
INTRAPULMONARY SMOOTH MUSCLE AND C0 2 w a y s previously c o n s t r i c t e d b y a n y of several different stimuli, including drugs or p u l m o n a r y artery occlusion. Ingram ( 1 9 7 5 ) , w h o separated t h e effects of changes in arterial CO2 and airway CO 2 in t h e dog, f o u n d t h a t decreases from n o r m a l airway CO2 increased lung resistance and t h a t , conversely, increases in arterial CO2 were directly related t o increases in resistance which were vagally m e d i a t e d .
Both King and Cowie ( 1 9 6 9 ) and Lewis ( 1 9 2 4 ) observed r e p e a t e d r h y t h m i c contractions of avian bronchial s m o o t h muscle in vivo and in vitro after t h e y applied e x c i t a t o r y drugs; t h e contractions were arrested b y a t r o p i n e . T h e s p o n t a n e o u s c o n t r a c t i o n s we observed occurred irregularly. Our results indicated t h a t t h e contractions could b e abolished b y v a g o t o m y b u t could n o t b e totally inhibited by atropine. T h u s , it is difficult t o ascertain w h e t h e r t h e s p o n t a n e o u s c o n t r a c t i o n s were neurally induced or resulted from s o m e intrinsic p r o p e r t y of t h e s m o o t h muscle.
ACKNOWLEDGMENTS We t h a n k Wade K u h l m a n n , Dean Klentz, Dalyn Wilson, and David A d a m s for their technical assistance and helpful advice.
REFERENCES Astin, T. W., G. R. Barer, J. W. Shaw, and P. M. Warren, 1973. The action of carbon dioxide on constricted airways. J. Physiol. (London) 2 3 5 : 6 0 7 623. Duckies, S. P., M. D. Rayner, and J. A. Nadel, 1974. Effects of C0 2 and pH on drug-induced contractions of airway smooth muscle. J. Pharm. Exp. Ther. 190:472-481. Fedde, M. R., R. N. Gatz, H. Slama, and P. Scheid, 1974a. Intrapulmonary C 0 2 receptors in the duck: I. Stimulus specificity. Respir. Physiol. 22:99— 114. Ingram, R. H., 1975. Effects of airway versus arterial C0 2 changes on lung mechanics in dogs. J. Appl. Physiol. 38:603-607. King, A. S., and A. F. Cowie, 1969. The functional anatomy of the bronchial muscle of the bird. J. Anat. 105:323-336. King, A. S., and V. Molony, 1971. The anatomy of respiration. Page 93—169 in Physiology and biochemistry of the domestic fowl. D. J. Bell and B. M. Freeman, ed. Academic Press, New York. Leitner, M.-M., and M. Roumy, 1974. Vagal afferent activities related to the respiratory cycle in the duck: Sensitivity to mechanical, chemical and electrical stimuli. Respir. Physiol. 22:41—56. Lewis, M. R., 1924. Spontaneous rhythmical contraction of muscles of the bronchial tubes and air sacs of the chick embryo. Amer. J. Physiol. 68:385— 388. Molony, V., W. Graf, and P. Scheid, 1976. Effects of C0 2 on pulmonary air flow resistance in the duck. Respir. Physiol. 26:333-349. Nadel, J. A., and J. G. Widdicombe, 1962. Effect of changes in blood gas tensions and carotid sinus pressure on tracheal volume and total lung resistance to flow. J. Physiol. (London) 163:13—33. Nisell, O. I., 1950. The action of oxygen and carbon dioxide in the bronchioles and vessels of the isolated perfused lung. Acta Physiol. Scand. 21:5—62. Ray, P. J., and M. R. Fedde, 1969. Responses to alterations in respiratory P o 2 and PC0 2 in the chicken. Respir. Physiol. 6:135-143. Severinghaus, J. W., E. W. Swenson, T. N. Finley, M. T. Lategola, and J. Williams, 1961. Unilateral hypoventilation produced in dogs by occluding one pulmonary artery. J. Appl. Physiol. 16:53—60.
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In our s t u d y , arterial CO2 was n o t monitored, so it was difficult t o estimate t h e i m m e d iate changes in t h e CO2 c o n c e n t r a t i o n of t h e blood a c c o m p a n y i n g changes in F i c o • However, it is reasonable t o assume t h a t t h e interaction of t h e t w o contrasting mechanisms — t h e direct action of F I C Q and t h e vagally mediated response t o arterial C 0 2 changes — c o n t r i b u t e d t o t h e wide variance in response of t h e parabronchial s m o o t h muscle (Fig. 3). Mechanism of the Smooth Muscle Responses. T h e response of t h e parabronchial s m o o t h muscle t o mechanical stimulation was n o t abolished b y v a g a t o m y ; t h a t suggests a localized intrinsic m e c h a n i s m , one n o t neurally mediated, was involved. Our observations of neurally induced contraction of t h e s m o o t h muscle during vagal stimulation agree with those of King and Cowie ( 1 9 6 9 ) and Leitner a n d R o u m y ( 1 9 7 4 ) . We further observed t h a t this response was blocked by a t r o p i n e , which c o m p e t e s with t h e excitat o r y t r a n s m i t t e r s u b s t a n c e , acetylcholine, for receptor sites a t p a r a s y m p a t h e t i c n e u r o m u s cular j u n c t i o n s .
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