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Autonomic innervation of the pulmonary vascular bed in a toad (Bufo marinus)
Comp. gen. Pharmac., I97x , 2~ 287-294. [Scientechnica (Publishers) Ltd.]
287
A U T O N O M I C I N N E R V A T I O N OF THE P U L M O N A R Y V A S C U L A R BED IN A TOAD
(BUFOMARINUS)
G. C A M P B E L L Department of Zoology, Melbourne University, Parkville, 3o52, Victoria, Australia
(Received 14 Jan., I97X) ABSTRACT i. Effects of nerve stimulation on pulmonary vascular perfusion pressure in salineperfused isolated lungs of Bufo marinus were studied. 2. Evidence is presented that contraction of pulmonary visceral muscle may increase pulmonary vascular resistance. However, there is a separate musculature, as yet not located, of the vascular bed. 3. The pulmonary vascular muscle is innervated by vagal cholinergic constrictor fibres and by sympathetic adrenergic dilator fibres. 4. Non-adrenergie, non-cholinergic fibres in the vagus nerve cause a fall in vascular resistance but this may be an indirect effect of relaxation of visceral muscle. 5. Two stages in the evolution of the autonomic nervous system are proposed. I n m a m m a l s the vagus nerve distributes a u t o n o m i c p a t h w a y s with cholinergic postganglionic elements b o t h to c a r d i o v a s c u l a r a n d to visceral muscles. I n a m p h i b i a n s the vagus does not p r o v i d e a cholinergic innerv a t i o n o f visceral muscle in either the s t o m a c h or the lung ( C a m p b e l l , I969, I97X ). Alt h o u g h it is clearly established t h a t there is a cholinergic v a g a l i n n e r v a t i o n of the a m p h i b i a n h e a r t (Loewi, 192i ) it r e m a i n s to be seen w h e t h e r the vagi p r o v i d e cholinergic i n n e r v a t i o n of a n y other p o r t i o n of the cardiov a s c u l a r system. C o u v r e u r (x889) first showed t h a t stimulation o f the v a g o s y m p a t h e t i c t r u n k causes arrest o f the p u l m o n a r y circulation in frogs. L u c k h a r d t a n d Carlson (I 92 x) found t h a t t r e a t m e n t with a t r o p i n e p r e v e n t e d this vasoc o n t r i c t o r response, showing t h a t the nervefibres involved are cholinergic. T h e y further found t h a t s t i m u l a t i o n o f the cervical symp a t h e t i c c h a i n p r i o r to its j u n c t i o n with the vagus d i d not cause p u l m o n a r y vasoconstriction, i n d i c a t i n g t h a t the constrictor fibres a r e v a g a l in origin. I n contrast, F e d o t o w (1938) found p u l m o n a r y vasoconstrictor fibres b o t h in the vagus a n d in the symp a t h e t i c outflows. I n this p a p e r a n e x a m i n a tion is m a d e o f p u l m o n a r y v a s o m o t o r fibres in the vagus a n d cervical s y m p a t h e t i c nerve supplies to the lung in B. marinus. A b r i e f
a c c o u n t o f the results has been given elsew h e r e ( C a m p b e l l , i968 ). METHODS Toads (Bufo marinus) weighing xoo-2oo g. were brain- and spine-pithed and the lung on one side (with vagosympathetic trunk, intracranial vagus nerve-roots, and cervical sympathetic chain attached) was dissected free as described previously (Campbell, i97i ). Cannulae were tied into the pulmonary artery and vein and the lung vasculature was perfused with McKenzie's solution (see Campbell, Burnstock, and Wood, I964) at 3 I° C. A Watson-Marlowe roller pump was used to pump perfusate through the vessels at a constant rate. The lung was suspended in a I oo-ml, organ bath containing warmed, bubbled McKenzie's solution. Back-pressure from the lung vessels was recorded via a T-piece, inserted in the perfusion line close to the lung, by means of a Statham pressure transducer. In most of the experiments a cannula was tied into the short bronchial stem, the lung was filled with McKenzie's solution, and intrapulmonary pressure was recorded with a Statham pressure transducer. Records were displayed on a Gilson polygraph. A Grass Model 5 stimulator was used to provide 1-msecond squarewave pulses at the required voltage and frequency to platinum-ring electrodes, shielded in moulded plastic, through which the nerves were pulled. Drugs used were: acetylcholine chloride, (4-)amphetamine sulphate, adrenaline tartrate, bretylium tosylate, carbachol (carbamylcholine chloride), hyoscine hydrobromide, and metanephrine hydrochloride. Concentrations refer to these salts. All injections into the perfusion line were made in a volume of o.o 5 ml.
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RESULTS RESTING VASCULAR TONE W h e n p r e p a r a t i o n s were set up, the r a t e o f perfusion was a d j u s t e d so t h a t the m e a n perfusion pressure was b e t w e e n 20 a n d 3 ° cm. H~O. T h e flow-rate n e e d e d to achieve this pressure was in the o r d e r o f 2 ml. p e r m i n u t e . N o further a d j u s t m e n t s o f flow-rate were m a d e d u r i n g the e x p e r i m e n t , b u t the perfusion pressure rose to 4 ° cm. H~O or m o r e over the course o f 3 - 4 hours. T h e slow rise o f pressure p r o b a b l y represents a n increase in tone in the muscle controlling the vasculature, r a t h e r t h a n tissue o e d e m a , because the inh i b i t o r y v a s c u l a r responses described b e l o w
pressure (Fig. I A), a c c o m p a n i e d b y the pulmonary relaxation already reported (Campbell, I97I ). T h e pressor response was visible w i t h i n z-2 seconds o f the onset o f stimulation, whereas the p u l m o n a r y r e l a x a t i o n d i d n o t start until a t least 4 seconds h a d passed. Single pulses a p p l i e d to the nerves caused a n a p p r e c i a b l e b u t e p h e m e r a l rise in pressure in some p r e p a r a t i o n s . D u r i n g 3G-second periods o f s t i m u l a t i o n with I - 2 pulses p e r second it was c o m m o n for the perfusion pressure to rise b e y o n d I GO cm. H 2 0 (Fig. I B). W h e n stimul a t i o n was s t o p p e d the pressure d r o p p e d to the resting value over the course of I - 2 minutes in fresh p r e p a r a t i o n s . H o w e v e r , in
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Fro. i .--Isolated saline-perfused toad lung, to show effects of nerve stimulation on vascular perfusion pressure (PP) and intrapulmonary pressure (IP). In panel A stimulation of the intracranial roots of the vagus nerve (V R I, at the bar) with I pulse per second at 5 V. for 3o seconds causes lung relaxation and a rise in perfusion pressure. Panel B, from the same preparation, shows the response to stimulation of the vagal roots with 2 pulses per second (VR2, at the bar), indicating the strength of the pressor response obtainable. Note that the pressor response in A and B is followed by a depressor response. Panel C, from a different preparation at different perfusion pressure amplification, shows that stimulation of the cervical sympathetic chain (CS, at the bar) with 5 pulses per second at 5 V. for 3o seconds causes lung contraction and a fall in perfusion pressure. Time marker: I minute. Perfusion pressure calibration: ioo cm. H=O, on the left for A and B, on the right for C. were small or a b s e n t in fresh p r e p a r a t i o n s b u t b e c a m e well d e v e l o p e d as the perfusion pressure rose d u r i n g the e x p e r i m e n t . P r e p a r a t i o n s were set u p in b o t h s u m m e r a n d w i n t e r m o n t h s , b u t no differences in resting v a s c u l a r tone or in the responses to nerve s t i m u l a t i o n were noticed. T H E VAGUS NERVES
S t i m u l a t i o n of the i n t r a c r a n i a l roots o f the vagus nerve caused a m a r k e d rise in perfusion
p r e p a r a t i o n s perfused for a n h o u r or more, w h i c h h a d d e v e l o p e d a v a s c u l a r tone, the perfusion pressure fell below the resting v a l u e a n d then recovered d u r i n g the next 4 - 5 minutes (Fig. I A , B). T h e depressor response to v a g a l s t i m u l a t i o n was well m a r k e d at s t i m u l a t i o n frequencies as low as I pulse p e r second. W h e n hyoscine (6>< Io -7 g. p e r ml.) was a d d e d to the perfusion fluid, the pressor response to v a g a l s t i m u l a t i o n was r a p i d l y
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INNERVATION OF TOAD LUNG VASCULATURE
abolished. In those preparations with vascular tone the response was converted to a pure fall of perfusion pressure (Fig. 2A, B) having the same time course as the pulmonary inhibition. This result indicates that the nervefibres mediating the pressor response are cholinergic. Acetylcholine (o.ox-[ lag.) injected into the perfusion line caused a prompt
rise in perfusion pressure (Fig. 3 A) which was prevented by hyoscine. The vagal depressor responses did not decrease during perfusion with solution containing the adrenergic neuron-blocking drug bretylium (xo -6 g. per ml.; Fig. 2C). The inhibitory fibres involved are therefore not adrenergic. B Hyoscine
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C Bretylium
D Amphetamine
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Fro. 2.--Isolated saline-perfused toad lung, vascular perfusion pressure record. Panel A shows control responses to stimulation of the intracranial vagal roots (V R, at first bar) with [ pulse per second at 5 V. for 30 seconds, and of the cervical sympathetic chain (CS, at second bar) with io pulses per second at 8 V. for 30 seconds. The record, obtained soon after the preparation was set up, shows that sympathetic stimulation causes a barely perceptible depressor response. I n panel B the perfusion fluid contains hyoscine (6 × IO -7 g. per ml.). Note that the vagal pressor effect has been abolished, revealing a depressor effect. Note also that the resting perfnsion pressure has risen and that sympathetic stimulation now causes a clear depressor response. In panel C the perfnsion fluid also contains bretylium (10 -3 g. per ml.) and the response to sympathetic stimulation has been abolished; the response to vagal stimulation is unaffected. In panel D amphetamine (io -6 g. per ml.) has also been added to the perfusion fluid, causing the reappearance of the sympathetic depressor response. T i m e marker: I minute. Vertical calibration: IOO cm. H 2 0 , with o on the event marker line. B
I
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H20
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•10100 I
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0.05~g 0-5~g Fio. 3.--Isolated saline-pert'used toad lung, vascular perfusion pressure recold. I n A injection of acetylcholine (Ach, 0.0 5 pg., at the bar) into the perfnsion line causes a pressor response. In B injection of adrenaline (Ad, 0- 5 pg., at the bar) causes a depressor response. T i m e marker: I minute. Vertical calibration: 1oo cm. H~O at left for A and at right for B.
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CAMPBELL
THE CERVICAL SYMPATHETIC CHAIN
Stimulation of the cervical sympathetic chain between the first spinal nerve and the junction of the sympathetic with the vagus caused only depressor responses in all preparatlons cxccpt one, noted in the following section. In preparations with little or no vascular tonc, sympathetic stimulation had virtually no effect on perfusion pressure (Fig. 2A). When vascular tone had developed, stimulation of the sympathetic chain caused a fall in perfusion pressure (Fig. I C, 2 B). The sympathetic depressor response was usually m u c h less marked than the fall of pressure caused by vagal stimulation. As perfusion pressure rose during the course of an experiment, vagal deprcssor responscs bccame obvious before any vascular responsc to sympathetic stimulation could be detected (Fig. 2A). Even when vascular tone was well developed, the sympathetic chain had to be stimulated with 2- 3 pulses pcr second before a clear vasodepression could be elicited. The response was short-lived and the recovery was completc within 3o--60 seconds of the end of each stimulation period. Stimulation via the electrodes placed around the pulmonary artery had no effect on perfusion pressure. H y o s c i n e (IO -6 g. per ml.) did not affect the response to sympathetic stimulation, but the response was abolished by bretylium (IO -s g. per ml.; Fig. 2C). The blockade produced by bretylium was alleviated when a m p h e t a m i n e (xo -6 g. per ml.) was added to the perfusion fluid (Fig. 2 D), showing that the blockade was specific. The fibres causing the depressor/response are therefore adrenergic. In accordance with this observation it was found that injection of adrenaline (o.x-ioo lag.), the transmitter substance released by adrenergic nerves in anurans, caused a fall in perfusion pressure in most preparations (Fig. 3B). When vascular tone was absent, adrenaline was usually without effect. In about IO per cent of preparations it was found that small doses (o.I-o. 5 lag.) of adrenaline caused a small pressor response (Fig. 6), large doses causing the more normal depressor response.
Comp. gen. Pharmac.
INTERACTIONS BETWEEN VISCERAL VASCULAR RESPONSES
AND
Two considerations prompt an investigation of the relationship between visceral and vascular responses in the toad lung. First, it is claimed that the arteries and veins in the substance of the lung ofB. marinus do not have a smooth muscle component in their walls (Robinson, 1969). The large arteries and veins in the free margins of the alveolar septa lie in a connective-tissue sheath, which also surrounds bundles of smooth muscle running parallel to the vessels (Robinson, I965) , but it is not clear whether this muscle should be regarded as having visceral or vascular functions. Second, some apparent interactions between lung contractions and perfusion pressure changes were noted in the present study. For example, in two of the preparations studied spontaneous changes in intrapulmonary pressure appeared after about 3 hours of perfusion. Each spontaneous rise and fall of intrapulmonary pressure was associated with a rise and fall in perfusion pressure (Fig. 4A). In one of these preparations the sympathetic chain was stimulated, causing the expected rise in intrapulmonary pressure. The excitatory response of the pulmonary visceral muscle was, in this case, accompanied by a rise in perfusion pressures. Both responses were about as large as the spontaneous changes (Fig. 4B). Since in all other preparations stimulation of the sympathetic nerves caused a depressor response, the pressor response recorded in this one experiment seems most likely to have been an indirect effect on the vasculature produced by contraction of smooth muscles serving a visceral function. T o determine the degree of dependence between visceral and vascular responses of the lung, a number of drugs known to act on smooth muscle were injected in the hope of finding compounds with selective effects on the intrapulmonary pressure or the perfusion pressure. The following experiments show that changes in perfusion pressure can occur without changes in intrapulmonary pressure. Injections of acetylcholine caused a rise in both perfusion and lung pressure. However,
INNERVATION OF TOAD LUNG VASCULATURE
I 9 7 I, 2
the perfusion pressure started to increase within a b o u t 2 seconds of injection, whereas the l u n g did not c o n t r a c t u n t i l a b o u t 15
29I
sec6nds h a d passed, at which time the vascular response h a d a l r e a d y passed its m a x i m u m (Fig. 5A). A similar, b u t less
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FIG. 4.--Isolated saline-perfused toad lung showing spontaneous and stimulated changes in vascular perfusion pressure (PP) and intrapulmonary pressure (I V). Panel A shows spontaneous lung contractions which seem to induce rises in perfusion pressure. In panel B, from the same preparation, stimulation of the cervical sympathetic chain (CS, at bar) with IO pulses per second at 8V. for 30 seconds causes pulmonary contraction and an abnormal pressor response, perhaps a secondary result of the contraction. Time marker: I minute. Perfusion pressure calibration: IOO cm. H=O.
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FIo. 5.--Isolated saline-perfused toad lung, showing independence of changes in vascular perfusion pressure ( PP) and intrapulmonary pressure (I P). In A and g injections of acetylcholine (A c h, o.o 5 gg.) and earbachol (Cbch, 25 gg.) respectively cause a rise in perfusion pressure before causing lung contraction. In C and D lung contraction without an associated pressor effect is elicited by the 'Druckreflex' caused by injecting o'o5 ml. of distilled water (Dist) and by metanephrine (Net, iooo gg.) in a preparation without 'Druckreflex', respectively. The panels are from four different preparations. Time marker: I minute. Perfusion pressure calibration: IOO cm. I-I=O with o at the event marker line.
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Comp. gen. Pharmac.
CAMPBELL
clear-cut separation of responses was obtained with injections of carbachol; again the rise in perfusion pressure was already declining before the pulmonary response had reached its maximum (Fig. 5B). It therefore seems quite clear that contractions can occur which affect vascular perfusion but which do not change lung pressure. Two procedures caused a rise in intrapulmonary pressure without affecting perfusion pressure. In about half of the preparations used the ' Druckreflex' described
spontaneous and nerve-mediated changes. The reasons for the variable effects of pulmonary contraction on vascular perfusion are not clear. For instance, the relationship does not depend simply on the magnitude of the pulmonary contraction. In Fig. 6 are shown two excitatory responses to a low dose of adrenaline (o'5 lag.), obtained within 2o minutes of each other. In each trial adrenaline caused lung contraction, but the second response was nearly four times larger than the first. However, each trial caused a similar
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Fro. 6.--Isolated saline-perfused toad lung, showing changes in vascular perfusion pressure (PP) and intrapulmonary pressure (IP) caused by injections of adrenaline (Ad, o. 5 lag., at bars). The response in B was obtained 20 minutes after that in A. Note that although the lung contraction is four times greater in B than in A, the pressor responses are comparable in amplitude. While the fact that adrenaline causes a pressor response and not the normal depressor one suggests that the response is an indirect result of lung contraction, the relationship between the two responses is not direct. Time marker: x minute. Perfusion pressure calibration, xoo cm. H=O. by Brecht (I947) was seen, i.e., any brief rise in perfusion pressure (for instance, the pressure rise caused by the act of injection) caused a more prolonged rise in intrapulmonary pressure. The effect of the injection of o.i ml. of distilled water into the perfusion line of such a preparation is shown in Fig. 5 C. The injection caused a contraction of the lung but there was no concurrent change in perfusion pressure. In preparations which did not show the Druckrefiex, it was found that large doses of metanephrine caused a small contraction of the lung without inducing a pressor response (Fig. 5D). O n the other hand, contractions of lung visceral muscle do sometimes appear to cause an increase in perfusion pressure, as noted above for
small rise in perfusion pressure. In this preparation, a larger dose of adrenaline (5 gg.) caused the normal depressor response. DISCUSSION In the toad, Bufo marinus, stimulation of the vagus nerves causes a rise in pulmonary vascular perfusion pressure, mediated by cholinergic nerves, and a depressor response mediated by non-adrenergic nerves. Stimulation of the cervical sympathetic contribution to the vagosympathetic trunk causes typically a depressor response, mediated by adrenergic nerves. However, it has been shown that there can be interactions between visceral and vascular functions in the lung. While there is no evidence that contractions
I97I, 2
INNERVATION
OF TOAD LUNG VASCULATURE
primarily affecting the vasculature have any effect on intrapulmonary pressure, the results presented indicate that contraction of the lung does, in some circumstances, increase the pulmonary vascular resistance. It must, therefore, be determined whether the changes in perfusion pressure caused by nerve stimulation do or do not reflect the activity of smooth muscle with the specific function of regulating blood-flow. The vagal cholinergic pressor effect is clearly a specific vascular response. Vagal stimulation causes a rise in perfusion pressure before any change in intrapulmonary pressure occurs, and the visceral muscle response, when it does occur, is a relaxation. The vagal depressor response, mediated by fibres which are neither adrenergic nor cholinergic, is of uncertain origin. T h e similar time course of the fall of intrapulmonary pressure and the fall of perfusion pressure caused by vagal stimulation m a y suggest that only one muscle type, the visceral, is responding. O n the other hand, it could well be that the one type of nerve-fibre acts on both visceral and vascular muscle with a similar time course. Since there is, as yet, no other evidence that these non-classic vagat nerve-fibres innervate any part of the cardiovascular system in amphibians or mammals, it is best to leave the question open. The sympathetic adrenergic depressor response appears to be a specific response of vascular muscle. The sympathetic outflow causes only contraction of the lung (Campbell, I97X) and it would therefore be expected to have the passive effect of increasing pulmonary vascular resistance. This response was, in fact, seen in one preparation. The depressor effect must therefore be a vascular response. This interpretation is in accordance with the observation that adrenaline, which causes contraction of the lung when injected in low doses and relaxation when in high doses (Campbell, 197 x), usually caused a vasodepression throughout the dose range tested. The sympathetic depressor response is weak compared with the vagal depressor effect and it might be supposed that the adrenaline reaches the vascular muscle by ' o v e r f l o w ' from neuromuscular
293
junctions at other muscle sites. However, a histochemical study of the adrenergic innervation of the lung in B. marinus (McLean and Burnstock, i967) showed that blood-vessels in the lung are provided with a scanty plexus of varicose adrenergic nerve-fibres. The weakness of the responses observed does raise doubts as to the physiological significance of the sympathetic vascular innervation. T h e observation made by Fedotow (i938), that in about half of the animals tested sympathetic stimulation caused vasoconstriction in the lung of Rana esculenta, could not be repeated in the toad. Either there is a genuine species difference or, as seems likely, stimulating current spread from the region of the sympathetic chain to the vascoconstrictor fibres in the nearby vagus in Fedotow's experiments. The fact that some muscle in the toad lung does have a specific function in vascular regulation raises the problem of the location of the muscle. According to Robinson (i 969), only the major vessels at the root of the toad lung have a smooth muscle layer in the wall. The majority of the vessels in the lung wall are not associated with smooth muscle cells, while the larger vessels in the margins of the alveolar septa lie next to longitudinal bundles of smooth muscle. The site of the vagal vasopressor effect does not seem to be in the main pulmonary artery because, in a few observations made on blood-flow in the lung surface in situ, it was found that stimulation of the vagosympathetic trunk stopped blood-flow with the major arterial branches distended with blood. In addition, stimulation via electrodes placed around the pulmonary artery had no effect on perfusion pressure. Boren and Krahl (1965) have given a brief description of ' kinking ' of vessels in the frog lung during vagosympathetic stimulation, suggesting that there is contraction of the muscle bundles running parallel to the septal vessels. Thus the pressor response m a y reflect crimping rather than radial constriction of blood-vessels in the lung. The situation m a y be rather different in the lung of Rana catesbiana, where Maloney and Castle (I969) found smooth muscle cells in the walls of arteries less than I oo la in diameter.
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CAMPBELL
I n conclusion, this s t u d y has shown t h a t the v a g a l a u t o n o m i c outflow provides cholinergic nerve-fibres to at least one v a s c u l a r b e d in the toad. Thus, the only cholinergic fibres k n o w n to exist in the vagus nerve of the t o a d s u p p l y c a r d i o v a s c u l a r structures. T h e v a g a t s u p p l y to visceral muscle o f the lungs a n d s t o m a c h is d e v o i d o f cholinergic fibres ( C a m p b e l l , I969, I 9 7 I ) , a feature w h i c h is surprising in the light o f m a m m a l i a n studies. T h e cholinergic i n n e r v a t i o n of visceral muscle in these organs is s u p p l i e d b y the s y m p a t h e t i c outflow. These observations suggest t h a t the composition o f the vagus nerve has evolved in two steps ( C a m p b e l l , i968 ) . T h e p r i m i t i v e vagus nerve c o n t a i n e d cholinergic fibres w h i c h were l i m i t e d in their d i s t r i b u t i o n to the c a r d i o v a s c u l a r system. T h e v a g a l s u p p l y to visceral s m o o t h muscle, on the o t h e r h a n d , consisted exclusively o f n o n - a d r e n e r g i c i n h i b i t o r y nervefibres. A t this stage the cholinergic i n n e r v a tion o f visceral s m o o t h muscle was p r o v i d e d , a l o n g w i t h the a d r e n e r g i c i n n e r v a t i o n , b y the s y m p a t h e t i c system. This is the position as seen in m o d e r n a m p h i b i a n s . A t some e v o l u t i o n a r y stage higher t h a n the a m p h i b i a n stock, the s y m p a t h e t i c cholinergic i n n e r v a tion of visceral muscles has shifted r o s t r a l l y into the v a g a l outflow to p r o v i d e the p a t t e r n o b s e r v e d in m a m m a l s . ACKNOWLEDGEMENTS
This work was supported by a grant from the Australian Research Grants Committee. Bretylium tosylate was donated by Burroughs Wellcome and Co. (Aust.) Ltd. I would like to thank Mr. B. H. Dumsday for his comments on the manuscript. REFERENCES BOREN, H. G., and KRAHL, V. E. (I965), 'Ventilatory, neural and other factors influencing the pulmonary microcirculation of the frog ', Bibl. Anat. 7, 86-91. BRECHT, K. (X947), ' Ober die Wirkung elektrischer Reizung des Vasosympathicns auf die
glatte Muskulatur der Froschlunge und ihre Beeinflussung durch Ionen bei kunstlicher Durchstr6mung ', pflfigers Arch. ges. Physiol., 249,
99-I
x x.
CAMPBELL, G. (1968), ' T w o separate lines of evolution in the autonomic nervous system ', Proc. int. Un. physiol. Sci., 6, 148-I49. CAMPBELL, G. (1969), ' T h e autonomic innervation of the stomach of a toad (Bufo marinus) ', Comp. Biochem. Physiol., 3 x, 693-7o6. CAMPBELL, G. (I97I), ' Autonomic innervation of the lung musculature of a toad (Bufo marinus) ', Comp. Biochem. Physiol., 2, 281-286. CAMPBELL, G., BURNSTOCK, G., and WOOD, M. (1964) , ' A method for distinguishing between adrenergic and cholinergic excitatory innervation of smooth muscle ', Q. 07. exp. Physiol., 49, 268-276. COUVREUR, E. (I889) , 'Influence de l'excitation du pneumogastrique sur la circulation pulmonaire de la grenouille ', C.r. hebd. S(anc. Acad. Sci., IO9, 823-825. FEDOTOW, J. P. (x938), ' Zur Frage fiber die vasomotorische Lungeninnervation ', Pfliigers Arch. ges. Physiol., 23 o, 273-282. LOEWI, O. (I921), ' Uber humorale Obertragbarkeit der Herznervenwirkung ', Pfli~gers Arch. ges. Physiol., x89, 239-242. LUCgHARDT, A. B., and CARLSON, A. J. (i921), ' Studies on the visceral sensory nervous system. V I I I . On the presence of vasomotor fibers in the vagus nerve to the pulmonary vessels of the amphibian and the reptilian lung ', Am. 07. Physiol., 56, 72-112. McLEAN, J. R., and BURNSTOCK, G. (I967), ' Innervation of the lungs of the toad (Bufo marinus). II. Fluorescent histochemistry of catecholamines ', Comp. Biochem. Physiol., 22, 767-774 . MALONEY, J. E., and CASTLE, B. L. (x969) , ' Pressure-diameter relations of capillaries and small blood vessels in frog lung ', Respir. Physiol., 7, I50--I62ROBXNSON, P. M. (I965), ' Fine structure of the autonomic innervation of the smooth muscle of the toad lung ', aT. Anat. 9 9 , 948-949 • ROBINSON, P. M. (I969), 'Studies on the fine structure of autonomic innervation ', P h . D . Thesis, Melbourne University.
Key Word Index: Pulmonary vasculature, lung, innervation, toad, Bufo marinus, vagus, sympathetic.
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A U T O N O M I C I N N E R V A T I O N OF THE P U L M O N A R Y V A S C U L A R BED IN A TOAD
(BUFOMARINUS)
G. C A M P B E L L Department of Zoology, Melbourne University, Parkville, 3o52, Victoria, Australia
(Received 14 Jan., I97X) ABSTRACT i. Effects of nerve stimulation on pulmonary vascular perfusion pressure in salineperfused isolated lungs of Bufo marinus were studied. 2. Evidence is presented that contraction of pulmonary visceral muscle may increase pulmonary vascular resistance. However, there is a separate musculature, as yet not located, of the vascular bed. 3. The pulmonary vascular muscle is innervated by vagal cholinergic constrictor fibres and by sympathetic adrenergic dilator fibres. 4. Non-adrenergie, non-cholinergic fibres in the vagus nerve cause a fall in vascular resistance but this may be an indirect effect of relaxation of visceral muscle. 5. Two stages in the evolution of the autonomic nervous system are proposed. I n m a m m a l s the vagus nerve distributes a u t o n o m i c p a t h w a y s with cholinergic postganglionic elements b o t h to c a r d i o v a s c u l a r a n d to visceral muscles. I n a m p h i b i a n s the vagus does not p r o v i d e a cholinergic innerv a t i o n o f visceral muscle in either the s t o m a c h or the lung ( C a m p b e l l , I969, I97X ). Alt h o u g h it is clearly established t h a t there is a cholinergic v a g a l i n n e r v a t i o n of the a m p h i b i a n h e a r t (Loewi, 192i ) it r e m a i n s to be seen w h e t h e r the vagi p r o v i d e cholinergic i n n e r v a t i o n of a n y other p o r t i o n of the cardiov a s c u l a r system. C o u v r e u r (x889) first showed t h a t stimulation o f the v a g o s y m p a t h e t i c t r u n k causes arrest o f the p u l m o n a r y circulation in frogs. L u c k h a r d t a n d Carlson (I 92 x) found t h a t t r e a t m e n t with a t r o p i n e p r e v e n t e d this vasoc o n t r i c t o r response, showing t h a t the nervefibres involved are cholinergic. T h e y further found t h a t s t i m u l a t i o n o f the cervical symp a t h e t i c c h a i n p r i o r to its j u n c t i o n with the vagus d i d not cause p u l m o n a r y vasoconstriction, i n d i c a t i n g t h a t the constrictor fibres a r e v a g a l in origin. I n contrast, F e d o t o w (1938) found p u l m o n a r y vasoconstrictor fibres b o t h in the vagus a n d in the symp a t h e t i c outflows. I n this p a p e r a n e x a m i n a tion is m a d e o f p u l m o n a r y v a s o m o t o r fibres in the vagus a n d cervical s y m p a t h e t i c nerve supplies to the lung in B. marinus. A b r i e f
a c c o u n t o f the results has been given elsew h e r e ( C a m p b e l l , i968 ). METHODS Toads (Bufo marinus) weighing xoo-2oo g. were brain- and spine-pithed and the lung on one side (with vagosympathetic trunk, intracranial vagus nerve-roots, and cervical sympathetic chain attached) was dissected free as described previously (Campbell, i97i ). Cannulae were tied into the pulmonary artery and vein and the lung vasculature was perfused with McKenzie's solution (see Campbell, Burnstock, and Wood, I964) at 3 I° C. A Watson-Marlowe roller pump was used to pump perfusate through the vessels at a constant rate. The lung was suspended in a I oo-ml, organ bath containing warmed, bubbled McKenzie's solution. Back-pressure from the lung vessels was recorded via a T-piece, inserted in the perfusion line close to the lung, by means of a Statham pressure transducer. In most of the experiments a cannula was tied into the short bronchial stem, the lung was filled with McKenzie's solution, and intrapulmonary pressure was recorded with a Statham pressure transducer. Records were displayed on a Gilson polygraph. A Grass Model 5 stimulator was used to provide 1-msecond squarewave pulses at the required voltage and frequency to platinum-ring electrodes, shielded in moulded plastic, through which the nerves were pulled. Drugs used were: acetylcholine chloride, (4-)amphetamine sulphate, adrenaline tartrate, bretylium tosylate, carbachol (carbamylcholine chloride), hyoscine hydrobromide, and metanephrine hydrochloride. Concentrations refer to these salts. All injections into the perfusion line were made in a volume of o.o 5 ml.
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RESULTS RESTING VASCULAR TONE W h e n p r e p a r a t i o n s were set up, the r a t e o f perfusion was a d j u s t e d so t h a t the m e a n perfusion pressure was b e t w e e n 20 a n d 3 ° cm. H~O. T h e flow-rate n e e d e d to achieve this pressure was in the o r d e r o f 2 ml. p e r m i n u t e . N o further a d j u s t m e n t s o f flow-rate were m a d e d u r i n g the e x p e r i m e n t , b u t the perfusion pressure rose to 4 ° cm. H~O or m o r e over the course o f 3 - 4 hours. T h e slow rise o f pressure p r o b a b l y represents a n increase in tone in the muscle controlling the vasculature, r a t h e r t h a n tissue o e d e m a , because the inh i b i t o r y v a s c u l a r responses described b e l o w
pressure (Fig. I A), a c c o m p a n i e d b y the pulmonary relaxation already reported (Campbell, I97I ). T h e pressor response was visible w i t h i n z-2 seconds o f the onset o f stimulation, whereas the p u l m o n a r y r e l a x a t i o n d i d n o t start until a t least 4 seconds h a d passed. Single pulses a p p l i e d to the nerves caused a n a p p r e c i a b l e b u t e p h e m e r a l rise in pressure in some p r e p a r a t i o n s . D u r i n g 3G-second periods o f s t i m u l a t i o n with I - 2 pulses p e r second it was c o m m o n for the perfusion pressure to rise b e y o n d I GO cm. H 2 0 (Fig. I B). W h e n stimul a t i o n was s t o p p e d the pressure d r o p p e d to the resting value over the course of I - 2 minutes in fresh p r e p a r a t i o n s . H o w e v e r , in
A
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Fro. i .--Isolated saline-perfused toad lung, to show effects of nerve stimulation on vascular perfusion pressure (PP) and intrapulmonary pressure (IP). In panel A stimulation of the intracranial roots of the vagus nerve (V R I, at the bar) with I pulse per second at 5 V. for 3o seconds causes lung relaxation and a rise in perfusion pressure. Panel B, from the same preparation, shows the response to stimulation of the vagal roots with 2 pulses per second (VR2, at the bar), indicating the strength of the pressor response obtainable. Note that the pressor response in A and B is followed by a depressor response. Panel C, from a different preparation at different perfusion pressure amplification, shows that stimulation of the cervical sympathetic chain (CS, at the bar) with 5 pulses per second at 5 V. for 3o seconds causes lung contraction and a fall in perfusion pressure. Time marker: I minute. Perfusion pressure calibration: ioo cm. H=O, on the left for A and B, on the right for C. were small or a b s e n t in fresh p r e p a r a t i o n s b u t b e c a m e well d e v e l o p e d as the perfusion pressure rose d u r i n g the e x p e r i m e n t . P r e p a r a t i o n s were set u p in b o t h s u m m e r a n d w i n t e r m o n t h s , b u t no differences in resting v a s c u l a r tone or in the responses to nerve s t i m u l a t i o n were noticed. T H E VAGUS NERVES
S t i m u l a t i o n of the i n t r a c r a n i a l roots o f the vagus nerve caused a m a r k e d rise in perfusion
p r e p a r a t i o n s perfused for a n h o u r or more, w h i c h h a d d e v e l o p e d a v a s c u l a r tone, the perfusion pressure fell below the resting v a l u e a n d then recovered d u r i n g the next 4 - 5 minutes (Fig. I A , B). T h e depressor response to v a g a l s t i m u l a t i o n was well m a r k e d at s t i m u l a t i o n frequencies as low as I pulse p e r second. W h e n hyoscine (6>< Io -7 g. p e r ml.) was a d d e d to the perfusion fluid, the pressor response to v a g a l s t i m u l a t i o n was r a p i d l y
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INNERVATION OF TOAD LUNG VASCULATURE
abolished. In those preparations with vascular tone the response was converted to a pure fall of perfusion pressure (Fig. 2A, B) having the same time course as the pulmonary inhibition. This result indicates that the nervefibres mediating the pressor response are cholinergic. Acetylcholine (o.ox-[ lag.) injected into the perfusion line caused a prompt
rise in perfusion pressure (Fig. 3 A) which was prevented by hyoscine. The vagal depressor responses did not decrease during perfusion with solution containing the adrenergic neuron-blocking drug bretylium (xo -6 g. per ml.; Fig. 2C). The inhibitory fibres involved are therefore not adrenergic. B Hyoscine
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Fro. 2.--Isolated saline-perfused toad lung, vascular perfusion pressure record. Panel A shows control responses to stimulation of the intracranial vagal roots (V R, at first bar) with [ pulse per second at 5 V. for 30 seconds, and of the cervical sympathetic chain (CS, at second bar) with io pulses per second at 8 V. for 30 seconds. The record, obtained soon after the preparation was set up, shows that sympathetic stimulation causes a barely perceptible depressor response. I n panel B the perfusion fluid contains hyoscine (6 × IO -7 g. per ml.). Note that the vagal pressor effect has been abolished, revealing a depressor effect. Note also that the resting perfnsion pressure has risen and that sympathetic stimulation now causes a clear depressor response. In panel C the perfnsion fluid also contains bretylium (10 -3 g. per ml.) and the response to sympathetic stimulation has been abolished; the response to vagal stimulation is unaffected. In panel D amphetamine (io -6 g. per ml.) has also been added to the perfusion fluid, causing the reappearance of the sympathetic depressor response. T i m e marker: I minute. Vertical calibration: IOO cm. H 2 0 , with o on the event marker line. B
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0.05~g 0-5~g Fio. 3.--Isolated saline-pert'used toad lung, vascular perfusion pressure recold. I n A injection of acetylcholine (Ach, 0.0 5 pg., at the bar) into the perfnsion line causes a pressor response. In B injection of adrenaline (Ad, 0- 5 pg., at the bar) causes a depressor response. T i m e marker: I minute. Vertical calibration: 1oo cm. H~O at left for A and at right for B.
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THE CERVICAL SYMPATHETIC CHAIN
Stimulation of the cervical sympathetic chain between the first spinal nerve and the junction of the sympathetic with the vagus caused only depressor responses in all preparatlons cxccpt one, noted in the following section. In preparations with little or no vascular tonc, sympathetic stimulation had virtually no effect on perfusion pressure (Fig. 2A). When vascular tone had developed, stimulation of the sympathetic chain caused a fall in perfusion pressure (Fig. I C, 2 B). The sympathetic depressor response was usually m u c h less marked than the fall of pressure caused by vagal stimulation. As perfusion pressure rose during the course of an experiment, vagal deprcssor responscs bccame obvious before any vascular responsc to sympathetic stimulation could be detected (Fig. 2A). Even when vascular tone was well developed, the sympathetic chain had to be stimulated with 2- 3 pulses pcr second before a clear vasodepression could be elicited. The response was short-lived and the recovery was completc within 3o--60 seconds of the end of each stimulation period. Stimulation via the electrodes placed around the pulmonary artery had no effect on perfusion pressure. H y o s c i n e (IO -6 g. per ml.) did not affect the response to sympathetic stimulation, but the response was abolished by bretylium (IO -s g. per ml.; Fig. 2C). The blockade produced by bretylium was alleviated when a m p h e t a m i n e (xo -6 g. per ml.) was added to the perfusion fluid (Fig. 2 D), showing that the blockade was specific. The fibres causing the depressor/response are therefore adrenergic. In accordance with this observation it was found that injection of adrenaline (o.x-ioo lag.), the transmitter substance released by adrenergic nerves in anurans, caused a fall in perfusion pressure in most preparations (Fig. 3B). When vascular tone was absent, adrenaline was usually without effect. In about IO per cent of preparations it was found that small doses (o.I-o. 5 lag.) of adrenaline caused a small pressor response (Fig. 6), large doses causing the more normal depressor response.
Comp. gen. Pharmac.
INTERACTIONS BETWEEN VISCERAL VASCULAR RESPONSES
AND
Two considerations prompt an investigation of the relationship between visceral and vascular responses in the toad lung. First, it is claimed that the arteries and veins in the substance of the lung ofB. marinus do not have a smooth muscle component in their walls (Robinson, 1969). The large arteries and veins in the free margins of the alveolar septa lie in a connective-tissue sheath, which also surrounds bundles of smooth muscle running parallel to the vessels (Robinson, I965) , but it is not clear whether this muscle should be regarded as having visceral or vascular functions. Second, some apparent interactions between lung contractions and perfusion pressure changes were noted in the present study. For example, in two of the preparations studied spontaneous changes in intrapulmonary pressure appeared after about 3 hours of perfusion. Each spontaneous rise and fall of intrapulmonary pressure was associated with a rise and fall in perfusion pressure (Fig. 4A). In one of these preparations the sympathetic chain was stimulated, causing the expected rise in intrapulmonary pressure. The excitatory response of the pulmonary visceral muscle was, in this case, accompanied by a rise in perfusion pressures. Both responses were about as large as the spontaneous changes (Fig. 4B). Since in all other preparations stimulation of the sympathetic nerves caused a depressor response, the pressor response recorded in this one experiment seems most likely to have been an indirect effect on the vasculature produced by contraction of smooth muscles serving a visceral function. T o determine the degree of dependence between visceral and vascular responses of the lung, a number of drugs known to act on smooth muscle were injected in the hope of finding compounds with selective effects on the intrapulmonary pressure or the perfusion pressure. The following experiments show that changes in perfusion pressure can occur without changes in intrapulmonary pressure. Injections of acetylcholine caused a rise in both perfusion and lung pressure. However,
INNERVATION OF TOAD LUNG VASCULATURE
I 9 7 I, 2
the perfusion pressure started to increase within a b o u t 2 seconds of injection, whereas the l u n g did not c o n t r a c t u n t i l a b o u t 15
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sec6nds h a d passed, at which time the vascular response h a d a l r e a d y passed its m a x i m u m (Fig. 5A). A similar, b u t less
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FIG. 4.--Isolated saline-perfused toad lung showing spontaneous and stimulated changes in vascular perfusion pressure (PP) and intrapulmonary pressure (I V). Panel A shows spontaneous lung contractions which seem to induce rises in perfusion pressure. In panel B, from the same preparation, stimulation of the cervical sympathetic chain (CS, at bar) with IO pulses per second at 8V. for 30 seconds causes pulmonary contraction and an abnormal pressor response, perhaps a secondary result of the contraction. Time marker: I minute. Perfusion pressure calibration: IOO cm. H=O.
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FIo. 5.--Isolated saline-perfused toad lung, showing independence of changes in vascular perfusion pressure ( PP) and intrapulmonary pressure (I P). In A and g injections of acetylcholine (A c h, o.o 5 gg.) and earbachol (Cbch, 25 gg.) respectively cause a rise in perfusion pressure before causing lung contraction. In C and D lung contraction without an associated pressor effect is elicited by the 'Druckreflex' caused by injecting o'o5 ml. of distilled water (Dist) and by metanephrine (Net, iooo gg.) in a preparation without 'Druckreflex', respectively. The panels are from four different preparations. Time marker: I minute. Perfusion pressure calibration: IOO cm. I-I=O with o at the event marker line.
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clear-cut separation of responses was obtained with injections of carbachol; again the rise in perfusion pressure was already declining before the pulmonary response had reached its maximum (Fig. 5B). It therefore seems quite clear that contractions can occur which affect vascular perfusion but which do not change lung pressure. Two procedures caused a rise in intrapulmonary pressure without affecting perfusion pressure. In about half of the preparations used the ' Druckreflex' described
spontaneous and nerve-mediated changes. The reasons for the variable effects of pulmonary contraction on vascular perfusion are not clear. For instance, the relationship does not depend simply on the magnitude of the pulmonary contraction. In Fig. 6 are shown two excitatory responses to a low dose of adrenaline (o'5 lag.), obtained within 2o minutes of each other. In each trial adrenaline caused lung contraction, but the second response was nearly four times larger than the first. However, each trial caused a similar
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Fro. 6.--Isolated saline-perfused toad lung, showing changes in vascular perfusion pressure (PP) and intrapulmonary pressure (IP) caused by injections of adrenaline (Ad, o. 5 lag., at bars). The response in B was obtained 20 minutes after that in A. Note that although the lung contraction is four times greater in B than in A, the pressor responses are comparable in amplitude. While the fact that adrenaline causes a pressor response and not the normal depressor one suggests that the response is an indirect result of lung contraction, the relationship between the two responses is not direct. Time marker: x minute. Perfusion pressure calibration, xoo cm. H=O. by Brecht (I947) was seen, i.e., any brief rise in perfusion pressure (for instance, the pressure rise caused by the act of injection) caused a more prolonged rise in intrapulmonary pressure. The effect of the injection of o.i ml. of distilled water into the perfusion line of such a preparation is shown in Fig. 5 C. The injection caused a contraction of the lung but there was no concurrent change in perfusion pressure. In preparations which did not show the Druckrefiex, it was found that large doses of metanephrine caused a small contraction of the lung without inducing a pressor response (Fig. 5D). O n the other hand, contractions of lung visceral muscle do sometimes appear to cause an increase in perfusion pressure, as noted above for
small rise in perfusion pressure. In this preparation, a larger dose of adrenaline (5 gg.) caused the normal depressor response. DISCUSSION In the toad, Bufo marinus, stimulation of the vagus nerves causes a rise in pulmonary vascular perfusion pressure, mediated by cholinergic nerves, and a depressor response mediated by non-adrenergic nerves. Stimulation of the cervical sympathetic contribution to the vagosympathetic trunk causes typically a depressor response, mediated by adrenergic nerves. However, it has been shown that there can be interactions between visceral and vascular functions in the lung. While there is no evidence that contractions
I97I, 2
INNERVATION
OF TOAD LUNG VASCULATURE
primarily affecting the vasculature have any effect on intrapulmonary pressure, the results presented indicate that contraction of the lung does, in some circumstances, increase the pulmonary vascular resistance. It must, therefore, be determined whether the changes in perfusion pressure caused by nerve stimulation do or do not reflect the activity of smooth muscle with the specific function of regulating blood-flow. The vagal cholinergic pressor effect is clearly a specific vascular response. Vagal stimulation causes a rise in perfusion pressure before any change in intrapulmonary pressure occurs, and the visceral muscle response, when it does occur, is a relaxation. The vagal depressor response, mediated by fibres which are neither adrenergic nor cholinergic, is of uncertain origin. T h e similar time course of the fall of intrapulmonary pressure and the fall of perfusion pressure caused by vagal stimulation m a y suggest that only one muscle type, the visceral, is responding. O n the other hand, it could well be that the one type of nerve-fibre acts on both visceral and vascular muscle with a similar time course. Since there is, as yet, no other evidence that these non-classic vagat nerve-fibres innervate any part of the cardiovascular system in amphibians or mammals, it is best to leave the question open. The sympathetic adrenergic depressor response appears to be a specific response of vascular muscle. The sympathetic outflow causes only contraction of the lung (Campbell, I97X) and it would therefore be expected to have the passive effect of increasing pulmonary vascular resistance. This response was, in fact, seen in one preparation. The depressor effect must therefore be a vascular response. This interpretation is in accordance with the observation that adrenaline, which causes contraction of the lung when injected in low doses and relaxation when in high doses (Campbell, 197 x), usually caused a vasodepression throughout the dose range tested. The sympathetic depressor response is weak compared with the vagal depressor effect and it might be supposed that the adrenaline reaches the vascular muscle by ' o v e r f l o w ' from neuromuscular
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junctions at other muscle sites. However, a histochemical study of the adrenergic innervation of the lung in B. marinus (McLean and Burnstock, i967) showed that blood-vessels in the lung are provided with a scanty plexus of varicose adrenergic nerve-fibres. The weakness of the responses observed does raise doubts as to the physiological significance of the sympathetic vascular innervation. T h e observation made by Fedotow (i938), that in about half of the animals tested sympathetic stimulation caused vasoconstriction in the lung of Rana esculenta, could not be repeated in the toad. Either there is a genuine species difference or, as seems likely, stimulating current spread from the region of the sympathetic chain to the vascoconstrictor fibres in the nearby vagus in Fedotow's experiments. The fact that some muscle in the toad lung does have a specific function in vascular regulation raises the problem of the location of the muscle. According to Robinson (i 969), only the major vessels at the root of the toad lung have a smooth muscle layer in the wall. The majority of the vessels in the lung wall are not associated with smooth muscle cells, while the larger vessels in the margins of the alveolar septa lie next to longitudinal bundles of smooth muscle. The site of the vagal vasopressor effect does not seem to be in the main pulmonary artery because, in a few observations made on blood-flow in the lung surface in situ, it was found that stimulation of the vagosympathetic trunk stopped blood-flow with the major arterial branches distended with blood. In addition, stimulation via electrodes placed around the pulmonary artery had no effect on perfusion pressure. Boren and Krahl (1965) have given a brief description of ' kinking ' of vessels in the frog lung during vagosympathetic stimulation, suggesting that there is contraction of the muscle bundles running parallel to the septal vessels. Thus the pressor response m a y reflect crimping rather than radial constriction of blood-vessels in the lung. The situation m a y be rather different in the lung of Rana catesbiana, where Maloney and Castle (I969) found smooth muscle cells in the walls of arteries less than I oo la in diameter.
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I n conclusion, this s t u d y has shown t h a t the v a g a l a u t o n o m i c outflow provides cholinergic nerve-fibres to at least one v a s c u l a r b e d in the toad. Thus, the only cholinergic fibres k n o w n to exist in the vagus nerve of the t o a d s u p p l y c a r d i o v a s c u l a r structures. T h e v a g a t s u p p l y to visceral muscle o f the lungs a n d s t o m a c h is d e v o i d o f cholinergic fibres ( C a m p b e l l , I969, I 9 7 I ) , a feature w h i c h is surprising in the light o f m a m m a l i a n studies. T h e cholinergic i n n e r v a t i o n of visceral muscle in these organs is s u p p l i e d b y the s y m p a t h e t i c outflow. These observations suggest t h a t the composition o f the vagus nerve has evolved in two steps ( C a m p b e l l , i968 ) . T h e p r i m i t i v e vagus nerve c o n t a i n e d cholinergic fibres w h i c h were l i m i t e d in their d i s t r i b u t i o n to the c a r d i o v a s c u l a r system. T h e v a g a l s u p p l y to visceral s m o o t h muscle, on the o t h e r h a n d , consisted exclusively o f n o n - a d r e n e r g i c i n h i b i t o r y nervefibres. A t this stage the cholinergic i n n e r v a tion o f visceral s m o o t h muscle was p r o v i d e d , a l o n g w i t h the a d r e n e r g i c i n n e r v a t i o n , b y the s y m p a t h e t i c system. This is the position as seen in m o d e r n a m p h i b i a n s . A t some e v o l u t i o n a r y stage higher t h a n the a m p h i b i a n stock, the s y m p a t h e t i c cholinergic i n n e r v a tion of visceral muscles has shifted r o s t r a l l y into the v a g a l outflow to p r o v i d e the p a t t e r n o b s e r v e d in m a m m a l s . ACKNOWLEDGEMENTS
This work was supported by a grant from the Australian Research Grants Committee. Bretylium tosylate was donated by Burroughs Wellcome and Co. (Aust.) Ltd. I would like to thank Mr. B. H. Dumsday for his comments on the manuscript. REFERENCES BOREN, H. G., and KRAHL, V. E. (I965), 'Ventilatory, neural and other factors influencing the pulmonary microcirculation of the frog ', Bibl. Anat. 7, 86-91. BRECHT, K. (X947), ' Ober die Wirkung elektrischer Reizung des Vasosympathicns auf die
glatte Muskulatur der Froschlunge und ihre Beeinflussung durch Ionen bei kunstlicher Durchstr6mung ', pflfigers Arch. ges. Physiol., 249,
99-I
x x.
CAMPBELL, G. (1968), ' T w o separate lines of evolution in the autonomic nervous system ', Proc. int. Un. physiol. Sci., 6, 148-I49. CAMPBELL, G. (1969), ' T h e autonomic innervation of the stomach of a toad (Bufo marinus) ', Comp. Biochem. Physiol., 3 x, 693-7o6. CAMPBELL, G. (I97I), ' Autonomic innervation of the lung musculature of a toad (Bufo marinus) ', Comp. Biochem. Physiol., 2, 281-286. CAMPBELL, G., BURNSTOCK, G., and WOOD, M. (1964) , ' A method for distinguishing between adrenergic and cholinergic excitatory innervation of smooth muscle ', Q. 07. exp. Physiol., 49, 268-276. COUVREUR, E. (I889) , 'Influence de l'excitation du pneumogastrique sur la circulation pulmonaire de la grenouille ', C.r. hebd. S(anc. Acad. Sci., IO9, 823-825. FEDOTOW, J. P. (x938), ' Zur Frage fiber die vasomotorische Lungeninnervation ', Pfliigers Arch. ges. Physiol., 23 o, 273-282. LOEWI, O. (I921), ' Uber humorale Obertragbarkeit der Herznervenwirkung ', Pfli~gers Arch. ges. Physiol., x89, 239-242. LUCgHARDT, A. B., and CARLSON, A. J. (i921), ' Studies on the visceral sensory nervous system. V I I I . On the presence of vasomotor fibers in the vagus nerve to the pulmonary vessels of the amphibian and the reptilian lung ', Am. 07. Physiol., 56, 72-112. McLEAN, J. R., and BURNSTOCK, G. (I967), ' Innervation of the lungs of the toad (Bufo marinus). II. Fluorescent histochemistry of catecholamines ', Comp. Biochem. Physiol., 22, 767-774 . MALONEY, J. E., and CASTLE, B. L. (x969) , ' Pressure-diameter relations of capillaries and small blood vessels in frog lung ', Respir. Physiol., 7, I50--I62ROBXNSON, P. M. (I965), ' Fine structure of the autonomic innervation of the smooth muscle of the toad lung ', aT. Anat. 9 9 , 948-949 • ROBINSON, P. M. (I969), 'Studies on the fine structure of autonomic innervation ', P h . D . Thesis, Melbourne University.
Key Word Index: Pulmonary vasculature, lung, innervation, toad, Bufo marinus, vagus, sympathetic.