- Email: [email protected]
Autonomic vasomotor innervation in the gas gland of the swimbladder of a teleost (Gadus Morhua)
Comp. gen. Pharmac., I972 , 3, 371-375 • [Scientechnica (Publishers) Ltd.]
371
A U T O N O M I C V A S O M O T O R I N N E R V A T I O N IN THE GAS GLAND OF THE SWIMBLADDER OF A TELEOST
(GADUS MORHUA) STEFAN NILSSON Department of Zoophysiology, University of G6teborg, Sweden
(Received6 April, I972 ) ABSTRACT i. The autonomic innervation of the gas gland vessels in the swimbladder of a teleost has been investigated by measurement of perfusion pressure at a constant flow rate. 2. Electrical stimulation of the splanchnic or vagus branches produced vasoconstriction, which could be diminished or abolished by adrenergic ct-receptor-blocking agents and bretylium and were usually diminished by atropine. 3. Acetylcholine or carbacholine in high concentrations produced a weak vasoconstriction, which could be abolished by atropine but was unchanged by ganglionic blocking agents, suggesting a direct action of acetylcholine on the vessels. 4. Adrenaline, noradrenaline, or phenylephrine all caused a marked vasocontriction, which was abolished by c~-adrenergic-blocking agents. Isoprenaline was without effect on the preparation. 5. It is suggested that the autonomic control of the vessels in the gas gland of the cod is mainly noradrenergic and possibly also to a smaller degree cholinergic, with the nervefibres running in the vagal and splanchnic branches.
GAs secretion in the s w i m b l a d d e r o f teleosts is p e r f o r m e d b y a specialized structure, the gas g l a n d , w h i c h in the cod is situated v e n t r a l l y in the a n t e r i o r p a r t of the s w i m b l a d d e r . This g l a n d receives b l o o d from a b r a n c h o f the celiac a r t e r y , a n d is i n n e r v a t e d b y b r a n c h e s o f the s p l a n c h n i c a n d vagus nerves (Stannius, I849 ; F/inge, I953, I966 ). T h e secretion o f gas from the gas g l a n d is blocked if the v a g a l b r a n c h e s a r e cut (Bohr, i 8 9 4 ; J a c o b s , I93 o, I933; F~nge, I953, x966), a n d secretory fibres in the vagus have b e e n suggested (F/inge, i953). V a s o m o t o r fibres h a v e b e e n t h o u g h t to r u n in the s p l a n c h n i c nerve (F/inge, I953) a n d S t r a y - P e d e r s e n (197o) d e m o n s t r a t e d v a g a l vasoconstrictor fibres in the eel. A d r e n e r g i c fibres in the vagus o f the cod a p p e a r to r e g u l a t e gas r e s o r p t i o n b y a c t i n g on the oval s p h i n c t e r o f the s w i m b l a d d e r (Nilsson, 197I ). V a s o m o t o r events seem to affect the blood-flow t h r o u g h the gas g l a n d in different stages o f gas secretion, a n d this m a y be o f g r e a t i m p o r t a n c e in r e g u l a t i n g the gas secretion (F/inge, i953).
I n the present investigation the n a t u r e o f the a u t o n o m i c v a s o m o t o r i n n e r v a t i o n has b e e n studied. M A T E R I A L S AND M E T H O D S Cod (Gadus morhua), 4o-5ocm. long with a body-weight of 5oo-75 ° g., were used in the experiments. All experiments were carried out at a room temperature of about 2o ° C. The animals were killed by a blow on the head and opened laterally on the right side, exposing the nerves and vessels entering and leaving the swimbladder (Fig. I). The gas gland was perfused from a slow injector via a catheter inserted into the celiac artery and all vessels leaving the celiac artery, except the swimbladder artery, were ligated. Vasomotor events were registered as changes in the pressure of the perfusion fluid at constant flow rate. Pressure registrations were made via a T-piece by a Statham transducer connected to a GRASS Polygraph mod. 7- Drugs could be added into the perfusion stream via another T-piece from a second slow injector, thus allowing different drugs of constant concentration to flow into the artery. The volume of the drug solution mixing with the perfusion fluid per unit time was kept below I per cent of the total volume. In a few
372
NILSSON
experiments a io per cent mixture of Indian ink was injected into the swimbladder artery to examine the distribution of the perfusion fluid. The gas gland vessels were rimmed with saline, followed by the Indian ink mixture. In some cases noradrenaline was added to the saline and to the Indian ink. Stimulation of the splanchnic and vagus branches was carried out via two silver electrodes
" t
~;t ~
v.n.
FIG. I.--Gadus morhua: Nerves and blood vessels outside the swimbladder on the right side. The head kidney has been removed to expose the celiac ganglion, c.a., Celiac artery; e.g., celiac ganglion; p.v., portal vein; s.a., swimbladder artery; s.v., swimbladder vein; spkn., splanchnic nerve; v.n., right vagus nerve.
by a GRASS SD9 stimulator. The perfusion fluid consisted of the saline earlier described (Nilsson, I97I ) with the addition of 3 per cent R M I Dextran (M.W.--45,ooo) (Pharmacia) to avoid oedema. The following drugs were used: acetylcholine chloride, L-adrenaline bitartrate, atropine sulphate, carbacholine chloride, hexamethonium bromide, DL-isoprenaline hydrochloride, mecamylamine hydrochloride, L-noradrenaline bitartrate, phenoxybenzamine hydrochloride, L-phenylephrine hydrochloride, propranolol hydrochloride, tyramine hydrochloride, yohimbine hydrochloride. Concentrations of these salts are expressed in g. per ml. RESULTS T o o b t a i n a perfusion pressure of 15-2o cm. H 2 0 , r o u g h l y c o r r e s p o n d i n g to the n o r m a l pressure in the cod celiac artery, a flow rate of
Comp. gen. Pharmac.
a b o u t 0. 3 ml. p e r m i n u t e was m a i n t a i n e d from the perfusion a p p a r a t u s . Before a n d after the e n t r a n c e o f the swimb l a d d e r the s w i m b l a d d e r a r t e r y branches, a n d some of the b l o o d is d i r e c t e d to the i n n e r surface of the s w i m b l a d d e r . W h e n the swimb l a d d e r is perfused in the described m a n n e r , the flow is r e g u l a t e d b y v a s o m o t o r activity, not only in the gas g l a n d b u t also in the rest o f the mucosa. I n three e x p e r i m e n t s the b l a d d e r cavity was o p e n e d d u r i n g perfusion a n d the larger vessels of the muscularis mucosae were ligated. T h i s caused a h a r d l y visible rise in perfusion pressure, i n d i c a t i n g t h a t all registra b l e v a s c u l a r events were t a k i n g place w i t h i n the gas gland. E L E C T R I C A L S T I M U L A T I O N OF T H E N E R V E S
Electrical s t i m u l a t i o n o f the s p l a n c h n i c nerve or either of the v a g a l b r a n c h e s with 2 o - 4 o pulses p e r second of 2-IO mseconds d u r a t i o n i n v a r i a b l y caused a vasoconstriction w i t h a n increased perfusion pressure o f 5 - I 0 cm. H 2 0 (Fig. 2A, B). T h e nerves could be s t i m u l a t e d a n y w h e r e along their courses, a n d were usually s t i m u l a t e d a few cm. outside the s w i m b l a d d e r . T h e effects of stimulation were diminished, or sometimes abolished, if d i b e n a m i n e (5 × I ° - 6 g. p e r ml.), y o h i m bine (5NIo-6g. p e r ml.), or b r e t y l i u m ( i o -6 g. p e r ml.) (Fig. 2B) was present in the perfusion fluid. T h e responses were usually diminished, a l t h o u g h never abolished, b y a t r o p i n e ( I o - S g . p e r ml.) (Fig. 2A) or p h e n o x y b e n z a m i n e (IO -5 g. p e r ml.), while hexamethonium (IO-4 g. p e r ml.), or m e c a m y l a m i n e ( I o - 4 g . p e r ml.), or the [3-adrenergic-blocking agent, p r o p r a n o l o l ( i o -5 g. p e r ml.), d i d not seem to alter the responses to electrical stimulation. T h e r e seemed to be no difference in responses b e t w e e n the splanchnic a n d vagus nerves. EFFECTS OF DRUGS A c e t y l c h o l i n e (5 × I o - 5-2 X i o -- 4 g. p e r ml.) (Fig. 3 B) or c a r b a c h o l i n e (5X I o - 5 - 2 × Io -4 g. p e r ml.) h a d w e a k vasoconstrictory effects, while lower c o n c e n t r a t i o n s were w i t h o u t effects. T h e responses to these drugs were abolished b y a t r o p i n e ( 5 × I O - 6 g • per ml.), while the ganglionic b l o c k i n g agents,
373
GAS GLAND INNERVATION OF TELEOST
I972, 3
hexamethonium ( I O - 4 g. per ml.) or m e c a m y l a m i n e (xo -4 g. per ml.) were without effects,
by dibenamine (5 × x ° - s g. per ml.) or p h e n o x y b e n z a m i n e ( 5 x lO -6 g. per ml.), while bretylium (Io -e g. per ml.) (Fig. 2B),
cm H20 30
t
A! ~ ~ '
IO
V
S Air 10
V
S
cm H20
20
IO
-
I
-
!
S
Bre 1
S
A 2
5 min
FIo. ~.--Gadus morhua: A, B, Effects of nerve stimulation and drugs on the blood-vessels of the gas gland. Atr, Atropine; Bre, bretylium; v, stimulation of the right vagus for 4 ° seconds; s, stimulation of the splanchnic nerve for 4 ° seconds. Numbers refer to the concentration of the drugs in lag. per ml. crn H20 30 A
I0
NA 1.2
cm H20
20 I
I0
ACh 200
II
NA 2
Stain
FIG.
3.--Gadus morhua: A,B, Effects of drugs on the gas gland vessels. Ach, Acetylcholine; NA, noradrenaline. Numbers refer to the concentration of the drugs in lag. per ml.
Vasoconstriction was seen with adrenaline ( 5 × I o - S - I o - S g . per ml.) (Fig. 2B), noradrenaline (5 x I o - S - [ o -5 g. per ml.) (Fig. 3A, B), and phenylephrine ( 1 o - 6 - 1 o -5 g. per ml.). T h e vasomotor effects of a n y of these amines (5 × I o - n g. per ml.) could be blocked
atropine ( I o - S g . per ml.), or propranolol (io -5 g. per ml.) did not visibly affect the responses. T y r a m i n e (xo -n g. per ml.) or isoprenaline ( i o - V - i o -5 g. per ml.) had no visible effects on the vessels of the gas gland.
NILSSON
374
INDIAN INK INJECTION Indian ink injected into the gas gland artery of a normal animal was directed into almost all parts of the gas gland (Fig. 4a). Sometimes small areas towards the edges of the gland remained unperfused by the solution. I f noradrenaline (lO -6 g. per ml.) present in the
O.
FIG.
Comp. gen. Pharmac.
drugs must, therefore, be thought to act on the vessels directly and not on the ganglia. The fact that atropine diminishes the responses to nerve stimulation could indicate presence of cholinergic vasomotor fibres, although the sensitivity to acetylcholine is very low. The large amounts of aeetylcholineesterase found
b.
4.--Gadus morhua: a, Gas gland injected with Indian ink. b, Gas gland injected with io -6 g. per
ml. noradrenaline followed by Indian ink. Dotted areas indicate the distribution of Indian ink; drawings from photographs. perfusion medium was allowed to pass the gland before the Indian ink was injected, large areas of the gland remained unperfused and appeared to be more or less closed off from the path of the perfusion fluid (Fig. 4b). DISCUSSION From the results it is evident that vasoconstrictor fibres to the gas gland vessels run in the splanchnic and the vagal branches. T h e vasoconstrictor innervation m a y act on precapillary sphincters in the rete mirabilae, more or less closing off"areas of the gas gland, as seen from the Indian ink injections with noradrenaline. Campbell ( 1971 ) demonstrated cholinergic vasoconstrictor fibres in the vasculature of the toad lung, and K i r b y and Burnstock (1969) concluded that the tonus of large arteries of lower vertebrates is regulated by both cholinergic and adrenergic fibres. In the present work acetylcholine and carbacholine administered in relatively high doses produced weak vasoconstriction, the effect being abolished by atropine but unchanged by ganglionic blocking agents. These
in the swimbladder (Augustinsson and F~inge, 195I ) m a y be localized to such cholinergic fibres and to fibres directly innervating the gas gland cells. Sectioning the vagus nerve blocks all secretion of gas from the gas gland (Bohr, i894; Jacobs, I93 o, I933) , and secretion is also inhibited by atropine (F~inge, 1953). The secretory fibres of the vagus are therefore thought to be cholinergic. Adrenaline, noradrenaline, and phenylephrine cause vasoconstriction, while isoprenaline lacks effects. This, together with the fact that adrenergic or-receptor-blocking agents inhibit the effects of these drugs and the effects of nerve stimulation, suggests an ct-adrenergic mechanism as being responsible for the vasoconstriction. Such a mechanism appears to be responsible for systemic vasoconstriction in teleosts (Randall and Stevens, 1967). The results with bretylium also support the presence of adrenergic vasoconstrictor innervation from the splanchnic and the vagus branches. The failure of tyramine to produce vasomotor responses could depend on a too small release ofnoradrenaline from the nerves (Kirby and Burnstock, 1969).
z972, 3
GAS GLAND INNERVATIONOF TELEOST
Sufficient a m o u n t s o f n o r a d r e n a l i n e to c o r r e s p o n d to a n a d r e n e r g i c i n n e r v a t i o n o f the gas g l a n d were f o u n d b y y o n E u l e r a n d F ~ n g e ( I 9 6 I ) , a n d Fahl~n, F a l c k , a n d R o s e n g r e n (1965) d e t e c t e d fibres c o n t a i n i n g n o r a d r e n aline a m o n g the vessels o f the rete m i r a b i l a e in the gas g l a n d of the cod. A d r e n e r g i c v a g a l fibres to the muscularis m u c o s a e of the swimb l a d d e r have also been d e m o n s t r a t e d (Fringe, 1953; Nilsson, i 9 7 I ). N o o r d i n a r y g a n g l i a close to the swimb l a d d e r a p p e a r to be involved in the vasom o t o r regulation. T h e role o f the gas g l a n d g a n g l i o n o b s e r v e d b y F a h l 6 n a n d others (i 965) r e m a i n s u n k n o w n . N o v a s o d i l a t o r fibres were d e t e c t e d in the p r e s e n t study, a n d the lack o f a c t i v i t y of isop r e n a l i n e agrees w i t h the findings of Burnstock a n d K i r b y (I968) on large a r t e r y strips from lower vertebrates, w h e r e i n h i b i t o r y receptors for c a t e c h o l a m i n e s a p p e a r to be absent. T h e v a s o d i l a t a t i o n caused b y i s o p r e n a l i n e in the eel s w i m b l a d d e r r e p o r t e d b y S t r a y - P e d e r s e n (I97O) m a y , as p o i n t e d out b y this a u t h o r , be d u e to r e l a x a t i o n o f the muscularis m u c o s a e a n d not d u e to v a s o m o t o r events. T h e strong v a s o d i l a t a t i o n , w h i c h can be observed d u r i n g gas secretion, could o c c u r b y l i b e r a t i o n of locally vasoactive substances from the gas g l a n d cells d u r i n g secretory a c t i v i t y (F~inge, 1953). T h e vessels o f the gas g l a n d in the cod swimbladder are conceivably regulated mainly b y n o r a d r e n e r g i c fibres a n d p e r h a p s , to some s m a l l e r degree, b y cholinergic fibres causing vasoconstriction. These fibres r u n in b o t h the vagus a n d the s p l a n c h n i c nerves, while fibres to the muscularis mucosae in the s w i m - b l a d d e r a p p e a r to be solely a d r e n e r g i c a n d r u n in the vagus nerve o n l y (Nilsson, I971). ACKNOWLEDGEMENTS This work was supported by the Lars Hierta Foundation. I thank Professor R. F~inge for valuable advice and discussions. I also thank Kristinebergs Zoologiska Station for the animal material, and MSD, AB Mekos, and Scanmeda AB for supplying drugs.
375
REFERENCES AUGUSTINSSON, K. B., and F£NGE, R. (195Q,
' Innervation and acetylcholine splitting activity in the airbladder of fishes ', Acta physiol, scand., 229 224-23 ° • BOnR, C. (1894), ' The influence of section of the vagus nerve on the disengagement of gases in the airbladder of fishes ', J. Physiol., Lond., 15, 494-5oo. BURNSTOCK, G., and KIRBY, S. (I968), 'Absence of inhibitory effects of catecholamines on lower vertebrate stlip preparations ', 07. Pharm. Pharmac., 20, 404-406. CAMPBELL, G. (I 97I), ' Autonomic innervation of the pulmonary vascular bed in a toad (Bufo marinus) ', Comp. gen. Pharmac., 2, 287-294. YON EULER, U. S., and F£NGE, R. (I961), ' Catecholamines in nerves and organs of Myxine glutinosa, Squalus acanthias and Gadus callarias ', Gen. comp. Endocr., x, I91-194. FAHLgN, G. B., FALCK, B., and ROSENGR~N, E. (I965), ' Monoamines in the swimbladder of Gadus callarias and Salmo #ideus ', Acta physiol. scand., 649 I i9-i26. F~NGE, R. (1953) , ' The mechanisms of gas transport in the euphysoclist swimbladder', Acta physiol, scand., 3o9 suppl, xio, x-i33. FXNOE, R. (I966), 'Physiology of the swimbladder ', Physiol. Rev., 46, 299-322JACOBS, W. (I930), ' Untel~uchungen zur Physiologie der Schwimmblase. I. Uber Gassekretion in der Schwimmblase von Physoklisten ', Z. vergl. Physiol., IX, 565. JACOBS, W. (I933) , ' Untersuchungen zur Physiologie der Schwimmblase. II. Volumregulation in der Schwimmblase der Flussbarsches ', Z-
vergl. Physiol., x8, I25. KIRBY, S., and BURNSTOCK,G. (1969), ' Comparative pharmacological studies of isolated spiral strips of large arteries from lower vertebrates ', Comp. Biochem. Physiol., 28, 3o7-32o. NILSSON, S. (I97I), ' Adrenergie innervation and drug responses of the oval sphincter in the swimbladder of the cod (Gadus morhua) ', Acta physiol, scand., 839 446-453. RANDALL, D. J., and STEVENS,E. D. (1967), ' The role of adrenergic receptors in cardiovascular changes associated with exercise in salmon ', Comp. Biochem. Physiol., 2t, 415-424 . STANNXt~S, H. (1849), Das Peripherische Nervensystem der Fische. Rostock I849. STRAY-PEDERSEN, S, (I97o), ' Vascular responses induced by drugs and by vagal stimulation in the swimbaldder of the eel (Anguilla vulgaris) ', Comp. gen. Pharmac., x, 358-364 .
Key Word Index: Teleost fish, autonomic nerves, vasomotor innervation, swimbladder, circulation, Gadus morhua.
371
A U T O N O M I C V A S O M O T O R I N N E R V A T I O N IN THE GAS GLAND OF THE SWIMBLADDER OF A TELEOST
(GADUS MORHUA) STEFAN NILSSON Department of Zoophysiology, University of G6teborg, Sweden
(Received6 April, I972 ) ABSTRACT i. The autonomic innervation of the gas gland vessels in the swimbladder of a teleost has been investigated by measurement of perfusion pressure at a constant flow rate. 2. Electrical stimulation of the splanchnic or vagus branches produced vasoconstriction, which could be diminished or abolished by adrenergic ct-receptor-blocking agents and bretylium and were usually diminished by atropine. 3. Acetylcholine or carbacholine in high concentrations produced a weak vasoconstriction, which could be abolished by atropine but was unchanged by ganglionic blocking agents, suggesting a direct action of acetylcholine on the vessels. 4. Adrenaline, noradrenaline, or phenylephrine all caused a marked vasocontriction, which was abolished by c~-adrenergic-blocking agents. Isoprenaline was without effect on the preparation. 5. It is suggested that the autonomic control of the vessels in the gas gland of the cod is mainly noradrenergic and possibly also to a smaller degree cholinergic, with the nervefibres running in the vagal and splanchnic branches.
GAs secretion in the s w i m b l a d d e r o f teleosts is p e r f o r m e d b y a specialized structure, the gas g l a n d , w h i c h in the cod is situated v e n t r a l l y in the a n t e r i o r p a r t of the s w i m b l a d d e r . This g l a n d receives b l o o d from a b r a n c h o f the celiac a r t e r y , a n d is i n n e r v a t e d b y b r a n c h e s o f the s p l a n c h n i c a n d vagus nerves (Stannius, I849 ; F/inge, I953, I966 ). T h e secretion o f gas from the gas g l a n d is blocked if the v a g a l b r a n c h e s a r e cut (Bohr, i 8 9 4 ; J a c o b s , I93 o, I933; F~nge, I953, x966), a n d secretory fibres in the vagus have b e e n suggested (F/inge, i953). V a s o m o t o r fibres h a v e b e e n t h o u g h t to r u n in the s p l a n c h n i c nerve (F/inge, I953) a n d S t r a y - P e d e r s e n (197o) d e m o n s t r a t e d v a g a l vasoconstrictor fibres in the eel. A d r e n e r g i c fibres in the vagus o f the cod a p p e a r to r e g u l a t e gas r e s o r p t i o n b y a c t i n g on the oval s p h i n c t e r o f the s w i m b l a d d e r (Nilsson, 197I ). V a s o m o t o r events seem to affect the blood-flow t h r o u g h the gas g l a n d in different stages o f gas secretion, a n d this m a y be o f g r e a t i m p o r t a n c e in r e g u l a t i n g the gas secretion (F/inge, i953).
I n the present investigation the n a t u r e o f the a u t o n o m i c v a s o m o t o r i n n e r v a t i o n has b e e n studied. M A T E R I A L S AND M E T H O D S Cod (Gadus morhua), 4o-5ocm. long with a body-weight of 5oo-75 ° g., were used in the experiments. All experiments were carried out at a room temperature of about 2o ° C. The animals were killed by a blow on the head and opened laterally on the right side, exposing the nerves and vessels entering and leaving the swimbladder (Fig. I). The gas gland was perfused from a slow injector via a catheter inserted into the celiac artery and all vessels leaving the celiac artery, except the swimbladder artery, were ligated. Vasomotor events were registered as changes in the pressure of the perfusion fluid at constant flow rate. Pressure registrations were made via a T-piece by a Statham transducer connected to a GRASS Polygraph mod. 7- Drugs could be added into the perfusion stream via another T-piece from a second slow injector, thus allowing different drugs of constant concentration to flow into the artery. The volume of the drug solution mixing with the perfusion fluid per unit time was kept below I per cent of the total volume. In a few
372
NILSSON
experiments a io per cent mixture of Indian ink was injected into the swimbladder artery to examine the distribution of the perfusion fluid. The gas gland vessels were rimmed with saline, followed by the Indian ink mixture. In some cases noradrenaline was added to the saline and to the Indian ink. Stimulation of the splanchnic and vagus branches was carried out via two silver electrodes
" t
~;t ~
v.n.
FIG. I.--Gadus morhua: Nerves and blood vessels outside the swimbladder on the right side. The head kidney has been removed to expose the celiac ganglion, c.a., Celiac artery; e.g., celiac ganglion; p.v., portal vein; s.a., swimbladder artery; s.v., swimbladder vein; spkn., splanchnic nerve; v.n., right vagus nerve.
by a GRASS SD9 stimulator. The perfusion fluid consisted of the saline earlier described (Nilsson, I97I ) with the addition of 3 per cent R M I Dextran (M.W.--45,ooo) (Pharmacia) to avoid oedema. The following drugs were used: acetylcholine chloride, L-adrenaline bitartrate, atropine sulphate, carbacholine chloride, hexamethonium bromide, DL-isoprenaline hydrochloride, mecamylamine hydrochloride, L-noradrenaline bitartrate, phenoxybenzamine hydrochloride, L-phenylephrine hydrochloride, propranolol hydrochloride, tyramine hydrochloride, yohimbine hydrochloride. Concentrations of these salts are expressed in g. per ml. RESULTS T o o b t a i n a perfusion pressure of 15-2o cm. H 2 0 , r o u g h l y c o r r e s p o n d i n g to the n o r m a l pressure in the cod celiac artery, a flow rate of
Comp. gen. Pharmac.
a b o u t 0. 3 ml. p e r m i n u t e was m a i n t a i n e d from the perfusion a p p a r a t u s . Before a n d after the e n t r a n c e o f the swimb l a d d e r the s w i m b l a d d e r a r t e r y branches, a n d some of the b l o o d is d i r e c t e d to the i n n e r surface of the s w i m b l a d d e r . W h e n the swimb l a d d e r is perfused in the described m a n n e r , the flow is r e g u l a t e d b y v a s o m o t o r activity, not only in the gas g l a n d b u t also in the rest o f the mucosa. I n three e x p e r i m e n t s the b l a d d e r cavity was o p e n e d d u r i n g perfusion a n d the larger vessels of the muscularis mucosae were ligated. T h i s caused a h a r d l y visible rise in perfusion pressure, i n d i c a t i n g t h a t all registra b l e v a s c u l a r events were t a k i n g place w i t h i n the gas gland. E L E C T R I C A L S T I M U L A T I O N OF T H E N E R V E S
Electrical s t i m u l a t i o n o f the s p l a n c h n i c nerve or either of the v a g a l b r a n c h e s with 2 o - 4 o pulses p e r second of 2-IO mseconds d u r a t i o n i n v a r i a b l y caused a vasoconstriction w i t h a n increased perfusion pressure o f 5 - I 0 cm. H 2 0 (Fig. 2A, B). T h e nerves could be s t i m u l a t e d a n y w h e r e along their courses, a n d were usually s t i m u l a t e d a few cm. outside the s w i m b l a d d e r . T h e effects of stimulation were diminished, or sometimes abolished, if d i b e n a m i n e (5 × I ° - 6 g. p e r ml.), y o h i m bine (5NIo-6g. p e r ml.), or b r e t y l i u m ( i o -6 g. p e r ml.) (Fig. 2B) was present in the perfusion fluid. T h e responses were usually diminished, a l t h o u g h never abolished, b y a t r o p i n e ( I o - S g . p e r ml.) (Fig. 2A) or p h e n o x y b e n z a m i n e (IO -5 g. p e r ml.), while hexamethonium (IO-4 g. p e r ml.), or m e c a m y l a m i n e ( I o - 4 g . p e r ml.), or the [3-adrenergic-blocking agent, p r o p r a n o l o l ( i o -5 g. p e r ml.), d i d not seem to alter the responses to electrical stimulation. T h e r e seemed to be no difference in responses b e t w e e n the splanchnic a n d vagus nerves. EFFECTS OF DRUGS A c e t y l c h o l i n e (5 × I o - 5-2 X i o -- 4 g. p e r ml.) (Fig. 3 B) or c a r b a c h o l i n e (5X I o - 5 - 2 × Io -4 g. p e r ml.) h a d w e a k vasoconstrictory effects, while lower c o n c e n t r a t i o n s were w i t h o u t effects. T h e responses to these drugs were abolished b y a t r o p i n e ( 5 × I O - 6 g • per ml.), while the ganglionic b l o c k i n g agents,
373
GAS GLAND INNERVATION OF TELEOST
I972, 3
hexamethonium ( I O - 4 g. per ml.) or m e c a m y l a m i n e (xo -4 g. per ml.) were without effects,
by dibenamine (5 × x ° - s g. per ml.) or p h e n o x y b e n z a m i n e ( 5 x lO -6 g. per ml.), while bretylium (Io -e g. per ml.) (Fig. 2B),
cm H20 30
t
A! ~ ~ '
IO
V
S Air 10
V
S
cm H20
20
IO
-
I
-
!
S
Bre 1
S
A 2
5 min
FIo. ~.--Gadus morhua: A, B, Effects of nerve stimulation and drugs on the blood-vessels of the gas gland. Atr, Atropine; Bre, bretylium; v, stimulation of the right vagus for 4 ° seconds; s, stimulation of the splanchnic nerve for 4 ° seconds. Numbers refer to the concentration of the drugs in lag. per ml. crn H20 30 A
I0
NA 1.2
cm H20
20 I
I0
ACh 200
II
NA 2
Stain
FIG.
3.--Gadus morhua: A,B, Effects of drugs on the gas gland vessels. Ach, Acetylcholine; NA, noradrenaline. Numbers refer to the concentration of the drugs in lag. per ml.
Vasoconstriction was seen with adrenaline ( 5 × I o - S - I o - S g . per ml.) (Fig. 2B), noradrenaline (5 x I o - S - [ o -5 g. per ml.) (Fig. 3A, B), and phenylephrine ( 1 o - 6 - 1 o -5 g. per ml.). T h e vasomotor effects of a n y of these amines (5 × I o - n g. per ml.) could be blocked
atropine ( I o - S g . per ml.), or propranolol (io -5 g. per ml.) did not visibly affect the responses. T y r a m i n e (xo -n g. per ml.) or isoprenaline ( i o - V - i o -5 g. per ml.) had no visible effects on the vessels of the gas gland.
NILSSON
374
INDIAN INK INJECTION Indian ink injected into the gas gland artery of a normal animal was directed into almost all parts of the gas gland (Fig. 4a). Sometimes small areas towards the edges of the gland remained unperfused by the solution. I f noradrenaline (lO -6 g. per ml.) present in the
O.
FIG.
Comp. gen. Pharmac.
drugs must, therefore, be thought to act on the vessels directly and not on the ganglia. The fact that atropine diminishes the responses to nerve stimulation could indicate presence of cholinergic vasomotor fibres, although the sensitivity to acetylcholine is very low. The large amounts of aeetylcholineesterase found
b.
4.--Gadus morhua: a, Gas gland injected with Indian ink. b, Gas gland injected with io -6 g. per
ml. noradrenaline followed by Indian ink. Dotted areas indicate the distribution of Indian ink; drawings from photographs. perfusion medium was allowed to pass the gland before the Indian ink was injected, large areas of the gland remained unperfused and appeared to be more or less closed off from the path of the perfusion fluid (Fig. 4b). DISCUSSION From the results it is evident that vasoconstrictor fibres to the gas gland vessels run in the splanchnic and the vagal branches. T h e vasoconstrictor innervation m a y act on precapillary sphincters in the rete mirabilae, more or less closing off"areas of the gas gland, as seen from the Indian ink injections with noradrenaline. Campbell ( 1971 ) demonstrated cholinergic vasoconstrictor fibres in the vasculature of the toad lung, and K i r b y and Burnstock (1969) concluded that the tonus of large arteries of lower vertebrates is regulated by both cholinergic and adrenergic fibres. In the present work acetylcholine and carbacholine administered in relatively high doses produced weak vasoconstriction, the effect being abolished by atropine but unchanged by ganglionic blocking agents. These
in the swimbladder (Augustinsson and F~inge, 195I ) m a y be localized to such cholinergic fibres and to fibres directly innervating the gas gland cells. Sectioning the vagus nerve blocks all secretion of gas from the gas gland (Bohr, i894; Jacobs, I93 o, I933) , and secretion is also inhibited by atropine (F~inge, 1953). The secretory fibres of the vagus are therefore thought to be cholinergic. Adrenaline, noradrenaline, and phenylephrine cause vasoconstriction, while isoprenaline lacks effects. This, together with the fact that adrenergic or-receptor-blocking agents inhibit the effects of these drugs and the effects of nerve stimulation, suggests an ct-adrenergic mechanism as being responsible for the vasoconstriction. Such a mechanism appears to be responsible for systemic vasoconstriction in teleosts (Randall and Stevens, 1967). The results with bretylium also support the presence of adrenergic vasoconstrictor innervation from the splanchnic and the vagus branches. The failure of tyramine to produce vasomotor responses could depend on a too small release ofnoradrenaline from the nerves (Kirby and Burnstock, 1969).
z972, 3
GAS GLAND INNERVATIONOF TELEOST
Sufficient a m o u n t s o f n o r a d r e n a l i n e to c o r r e s p o n d to a n a d r e n e r g i c i n n e r v a t i o n o f the gas g l a n d were f o u n d b y y o n E u l e r a n d F ~ n g e ( I 9 6 I ) , a n d Fahl~n, F a l c k , a n d R o s e n g r e n (1965) d e t e c t e d fibres c o n t a i n i n g n o r a d r e n aline a m o n g the vessels o f the rete m i r a b i l a e in the gas g l a n d of the cod. A d r e n e r g i c v a g a l fibres to the muscularis m u c o s a e of the swimb l a d d e r have also been d e m o n s t r a t e d (Fringe, 1953; Nilsson, i 9 7 I ). N o o r d i n a r y g a n g l i a close to the swimb l a d d e r a p p e a r to be involved in the vasom o t o r regulation. T h e role o f the gas g l a n d g a n g l i o n o b s e r v e d b y F a h l 6 n a n d others (i 965) r e m a i n s u n k n o w n . N o v a s o d i l a t o r fibres were d e t e c t e d in the p r e s e n t study, a n d the lack o f a c t i v i t y of isop r e n a l i n e agrees w i t h the findings of Burnstock a n d K i r b y (I968) on large a r t e r y strips from lower vertebrates, w h e r e i n h i b i t o r y receptors for c a t e c h o l a m i n e s a p p e a r to be absent. T h e v a s o d i l a t a t i o n caused b y i s o p r e n a l i n e in the eel s w i m b l a d d e r r e p o r t e d b y S t r a y - P e d e r s e n (I97O) m a y , as p o i n t e d out b y this a u t h o r , be d u e to r e l a x a t i o n o f the muscularis m u c o s a e a n d not d u e to v a s o m o t o r events. T h e strong v a s o d i l a t a t i o n , w h i c h can be observed d u r i n g gas secretion, could o c c u r b y l i b e r a t i o n of locally vasoactive substances from the gas g l a n d cells d u r i n g secretory a c t i v i t y (F~inge, 1953). T h e vessels o f the gas g l a n d in the cod swimbladder are conceivably regulated mainly b y n o r a d r e n e r g i c fibres a n d p e r h a p s , to some s m a l l e r degree, b y cholinergic fibres causing vasoconstriction. These fibres r u n in b o t h the vagus a n d the s p l a n c h n i c nerves, while fibres to the muscularis mucosae in the s w i m - b l a d d e r a p p e a r to be solely a d r e n e r g i c a n d r u n in the vagus nerve o n l y (Nilsson, I971). ACKNOWLEDGEMENTS This work was supported by the Lars Hierta Foundation. I thank Professor R. F~inge for valuable advice and discussions. I also thank Kristinebergs Zoologiska Station for the animal material, and MSD, AB Mekos, and Scanmeda AB for supplying drugs.
375
REFERENCES AUGUSTINSSON, K. B., and F£NGE, R. (195Q,
' Innervation and acetylcholine splitting activity in the airbladder of fishes ', Acta physiol, scand., 229 224-23 ° • BOnR, C. (1894), ' The influence of section of the vagus nerve on the disengagement of gases in the airbladder of fishes ', J. Physiol., Lond., 15, 494-5oo. BURNSTOCK, G., and KIRBY, S. (I968), 'Absence of inhibitory effects of catecholamines on lower vertebrate stlip preparations ', 07. Pharm. Pharmac., 20, 404-406. CAMPBELL, G. (I 97I), ' Autonomic innervation of the pulmonary vascular bed in a toad (Bufo marinus) ', Comp. gen. Pharmac., 2, 287-294. YON EULER, U. S., and F£NGE, R. (I961), ' Catecholamines in nerves and organs of Myxine glutinosa, Squalus acanthias and Gadus callarias ', Gen. comp. Endocr., x, I91-194. FAHLgN, G. B., FALCK, B., and ROSENGR~N, E. (I965), ' Monoamines in the swimbladder of Gadus callarias and Salmo #ideus ', Acta physiol. scand., 649 I i9-i26. F~NGE, R. (1953) , ' The mechanisms of gas transport in the euphysoclist swimbladder', Acta physiol, scand., 3o9 suppl, xio, x-i33. FXNOE, R. (I966), 'Physiology of the swimbladder ', Physiol. Rev., 46, 299-322JACOBS, W. (I930), ' Untel~uchungen zur Physiologie der Schwimmblase. I. Uber Gassekretion in der Schwimmblase von Physoklisten ', Z. vergl. Physiol., IX, 565. JACOBS, W. (I933) , ' Untersuchungen zur Physiologie der Schwimmblase. II. Volumregulation in der Schwimmblase der Flussbarsches ', Z-
vergl. Physiol., x8, I25. KIRBY, S., and BURNSTOCK,G. (1969), ' Comparative pharmacological studies of isolated spiral strips of large arteries from lower vertebrates ', Comp. Biochem. Physiol., 28, 3o7-32o. NILSSON, S. (I97I), ' Adrenergie innervation and drug responses of the oval sphincter in the swimbladder of the cod (Gadus morhua) ', Acta physiol, scand., 839 446-453. RANDALL, D. J., and STEVENS,E. D. (1967), ' The role of adrenergic receptors in cardiovascular changes associated with exercise in salmon ', Comp. Biochem. Physiol., 2t, 415-424 . STANNXt~S, H. (1849), Das Peripherische Nervensystem der Fische. Rostock I849. STRAY-PEDERSEN, S, (I97o), ' Vascular responses induced by drugs and by vagal stimulation in the swimbaldder of the eel (Anguilla vulgaris) ', Comp. gen. Pharmac., x, 358-364 .
Key Word Index: Teleost fish, autonomic nerves, vasomotor innervation, swimbladder, circulation, Gadus morhua.