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Evidence for a functional relationship between noradrenaline and neurohypophyseal peptides in the brainstem of rats
Brain Research, 422 (1987) 295-302 Elsevier
295
BRE 12920
Evidence for a functional relationship between noradrenaline and neurohypophyseal peptides in the brainstem of rats Mario Vallejo and Stafford L. Lightman Medical Unit, Charing Cross and Westminster Medical School, London ( U. K.) (Accepted 3 March 1987) Key words: Neurohypophyseal peptide; Noradrenaline; Peptide/amine interaction; Nucleus tractus soiitarius: Blood pressure; Brattleboro rat
The possibility of a functional relationship between noradrenaline and neurohypophyseal peptides in the control of cardiovascular function in the nucleus tractus solitarius of rats has been investigated. The hypotensive response to microinjections of noradrenaline (20 nmolI was abolished by simultaneous (but not prior) microinjections of [ArgS]vasopressin (AVP) and oxytocin at doses (0.9 pmoB which alone had no effects on cardiovascular parameters. AVP plus noradrenaline actually resulted in a transient pressor effect. Simul. taneous administration of [deamino-D-ArgS]vasopressm. a selective agonist of AVP V z receptors, did not modify the effect of noradrenaline, whereas the specific V 1 antagonist D-(CH2)5-Tyr(Me)-AVP partially decreased its intensity. When subthreshold doses of both AVP (0.9 pmol) and noradrenaline (10 pmol) were administered simultaneously, a pressor response was observed. In vasopressin-deficient Brattleboro rats. mlcroinjections of 0.9 pmol AVP had no effects, but a marked pressor response was observed after the administration of a higher dose (9 pmol). In parent strain Long-Evans rats. noradrenaline (20 nmol) also produced a hypotensive re~ sponse, but in Brattleboro rats microinjections of this amine elicited a marked pressor effect. In these rats. simultaneous administra~ tion of a subthreshold dose of AVP (0.9 pmol) reversed this response in such a way that a fall in blood pressure, similar totha~ observed in Long-Evans rats after injection of noradrenaline alone, was registered. These results provide evidence for a functional interaction between noradrenafine and neurohypophyseal peptides in the control of cardiovascular function in the brainstem.
INTRODUCTION The nucleus tractus solitarius (NTS) within the dorsomedial m e d u l l a contains a rich p o p u l a t i o n of noradrenergic neurons of the A 2 group 9'1°. which are a m a j o r source of catecholaminergic terminals in this area 22. This nucleus also receives a p r o j e c t i o n of neur o h y p o p h y s e a l p e p t i d e - c o n t a i n i n g fibres which originate from cell bodies in the h y p o t h a l a m i c parvocellular paraventricular nucleus 16'2°. It is now known that these peptides are synthesized in the h y p o t h a l a m u s and carried by axonal t r a n s p o r t to the NTS 27. In this area, neurophysin-containing varicosities have b e e n described in juxtaposition with n o r a d r e n a l i n e perikarya and dendrites is. Cardiovascular afferent fibres from pressure r e c e p t o r s in the thorax travel in the IX and X nerves to the NTS 14. where some vagal fibres
project to n o r a d r e n e r g i c n e u r o n e s of the A 2 group 2~ [ArgS]vasopressm (AVP) has been shown to alter noradrenaline t u r n o v e r in the NTS 23. a n d b o t h AVP and n o r a d r e n a l i n e alter cardiovascular function when injected into this nucleus 13'24"2s. The coexistence of amine and p e p t i d e neurotransmitters within the s a m e n e u r o n e s or their presence in pathways projecting to the same target cells strongly suggests the existence of interactions b e t w e e n them. Functional interactions have now been clearly described in the p e r i p h e r y s. In the brain, however, a relationship b e t w e e n amines and p e p t i d e s has b e e n reported in vitro, but physiologically functional interactions have proved difficult to d e m o n s t r a t e s . A l though the anatomical distribution of n e u r o h y p o p h y seal p e p t i d e s and amine n e u r o n s in the NTS suggests a possible interaction in the control of cardiovascular
Correspondence: M. Vallejo. Present address: MRC Molecular Neurobiology Unit. University of Cambridge Medical School. Hills Road, Cambridge CB2 2QH, U.K 0006-8993/87/$03.50 © 1987 Elsevier Science Publishers B.V. ~Biomedical Division ~
296 function, no studies have hitherto addressed this possibility. The present experiments were designed to look for evidence of any functional relationship between noradrenaline and neurohypophyseal peptides in the NTS of rats. MATERIALS AND METHODS
Male Sprague-Dawley rats (250-350 g) were anaesthetized with urethane (1.3 g/kg i.p.). The left common carotid artery was cannulated with polythene tubing and connected via a Statham P23 ID transducer to a Gould Thermal chart recorder for continuous measurement of blood pressure and heart rate. The animals were placed in a Kopf stereotaxic frame with the head flexed downwards to an angle of 45 °. After a midline incision through the skin, the dorsal neck muscles were retracted and the atlantooccipital membrane was incised in order to expose the dorsal surface of the brainstem. After 20-30 rain, an injection (0.2/.tl delivered over 10 s) was administered into the right NTS, using pulled glass micropipettes ( - 5 0 ~m in diameter) connected by plastic tubing to a 10-~1 Hamilton syringe driven by a microinfusion pump. The injections were placed at the level of the caudal tip of the area postrema, 0.3 mm lateral to the midline and 0.6 mm below the dorsal surface of the brainstem. Each rat received only one injection, unless otherwise indicated. In order to confirm correct placement, histological examination of frozen sections of the brains was carried out in rats injected with safranine into the same position at the end of the experiments. Sections were cut at 25 ~m, stained with thionine and compared to the atlas of Pellegrino et al. ~5, revealing that the injectate was distributed within the NTS area, with minimal diffusion into the adjacent dorsal motor nucleus of the vagus. Rats were injected with vehicle (0.9% saline), ( - ) noradrenaline bitartrate (Sigma, U.K.), [ArgS]vaso pressin (Cambridge Research Biochemicals, U.K.), oxytocin (Cambridge Research Biochemicals, U.K.), [deamino-D-ArgS]vasopressin (DDAVP) (Ferring, Malmo, Sweden) or D-(CHjs-Tyr(Me)AVP, a specific antagonist of V 1 receptors 11. All the peptides were administered at a dose of 0.9 pmol, except the V 1 antagonist, which was given at a dose of 9 pmol. Noradrenaline was administered at a dose of
either 10 pmol or 20 nmol. Some animals treated with saline, AVP or oxytocin received a further injection of noradrenaline (20 nmol) into the same position 10 min later. Simultaneous administration of noradrenaline and peptides was also performed. Noradrenaline was disolved in a solution of saline containing the appropriate amount of AVP, oxytocin, DDAVP or D(CH2)5-Tyr(Me)-AVP. The dual injection was administered unilaterally so that 0.9 pmol of each peptide (or 9 pmol in the case of the V~ antagonist) was coinjected with either 10 pmol or 20 nmol noradrenaline. One further group of rats received bilateral injections of either saline or D-(CH2)5-Tyr(Me)-AVP (9 pmol each side), and 10 min later a unilateral injection of noradrenaline (20 nmol). In another study, male AVP-deficient Brattleboro and parent strain Long-Evans rats (220-300 g) were subjected to the general procedure described above, and treated with single microinjections of noradrenaline (20 nmol) in the right NTS. In addition, some Brattleboro rats were given microinjections of either saline or AVP (0.9 or 9 pmol), Simultaneous administration of AVP (0.9 pmol) and noradrenaline (20 nmol) was also performed in these animals. All Brattleboro rats were infused throughout the experi-
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substances simultaneously (©, n = 6) into the nucleus tractus solitarius of Sprague-Dawley rats.
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Fig. 2. Haemodynamic effects observed 2 and 10 min after microinjections of 20 nmol noradrenaline alone (SALI or simultaneously with DDAVP, AVP, oxytocin (OXT), or the V 1 antagonist D-(CH2)5-Tyr(Me)-AVP (ANT) into the nucleus tractus solitarius of Sprague-Dawley rats. All the peptides were administered at a dose of 0.9 pmol, except the vasopressin antagonist which was given at a dose of 9 pmol. The number of rats is in parentheses; *P < 0.05 (Duncan's multiple-range test).
ments with 0.9% saline via a cannula inserted in the jugular vein, at a rate of 6 ml/h, in o r d e r to avoid dehydration. L o n g - E v a n s rats were also cannulated but not infused. The statistical analysis of cardiovascular changes within groups was c a r r i e d out using t h e one-way analysis of variance, unless otherwise indicated. Duncan's multiple-range test was used for multiple comparisons b e t w e e n groups. In this case statistical significance was accepted for P < 0.05.
Basal values of mean arterial pressure and heart rate in S p r a g u e - D a w l e y rats were 93.3 _+ 5.1 m m H g and 403.8 + 15 beats p e r min. respectively. N o significant alterations of cardiovascular p a r a m e t e r s were observed after microinjections of saline, AVP, oxytocin, D D A V P or o-(CH2)5-Tyr(Me)-AVP. A significant decrease ( P < 0.005) in both b l o o d pressure and h e a r t rate was h o w e v e r o b s e r v e d in response to microinjections of 20 nmol n o r a d r e n a l i n e (Fig. l). This hypotensive effect d e v e l o p e d rapidly and was maximal within 10 min. lasting for m o r e than 20 min. W h e n AVP and n o r a d r e n a l i n e were injected simultaneously, a transient but significant mcrease (P < 0.02. Student's t-test) in b l o o d pressure was observed (Fig. 1). This p r e s s o r response only lasted 5 min, following which m e a n arterial p r e s s u r e r e t u r n e d to basal values without further changes. A small decrease in heart rate was seen, but this did not achieve statistical significance (Fig. 1). Simultaneous administration of oxytocin a n d n o r a d r e n a l i n e p r o d u c e d no appreciable changes in cardiovascular p a r a m e t e r s (Fig. 2). A m a r k e d decrease in both mean arterial pressure and h e a r t rate ( P < 0.005) was o b s e r v e d after simultaneous administration of D D A V P and noradrenaline, and this r e s p o n s e was not significantly different from that o b s e r v e d when the a m i n e was injected alone (Fig. 2). A significant decrease (P < 0.005) in m e a n arterial pressure was also o b s e r v e d after simultaneous administration of noradrenatine and D-(CH2)5-Tyr(Me)-AVP. H o w e v e r , the maxim u m fall, o b s e r v e d 10 min after the injection, was significantly less than that o b s e r v e d when noradren-
TABLE I Haemodynamic effects of noradrenaline microinjections into the nucleus tractus solitarius of rats, after local pre-treatment with saline, AVP or oxytocin
Noradrenaline (20 nmol) was administered 10 rain after microinjection of saline, AVP (0.9 pmol) or oxytocin (0.9 pmol) into the same place. Values represent changes from mean arterial pressure just before the injection of noradrenaline, and are expressed as mean + S.E.M. of 6 experiments. *P < 0.05, one-way analysis of variance; tp < 0.02, ~P < 0~005. Time after noradrenaline
A Mean arterial pressure (mm Fig)
d Heart rate (beats/rain)
injection (min)
Saline/ noradrenaline*
AVP/ noradrenaline*
Oxytocin/ noradrenaline ~
Saline/ noradrenaline ~
AVP/ noradrenalme*
Oxytocin/ noradrenaline
2 5 10 15
-4.5±3.2 -13±3.7 -22±3.2 -~±3.6
- 5±2.4 -15.5±3.6 -~.5±4.5 -18.6±4.2
- 3.6±5,6 -17.5±2.5 -I8.6±4.4 -13.6±4.4
-38±6.5 -~±9.9 -7±11;1 -70±10.3
-65 -78 -88 -91
-51 "61 -70 -66
+ + ± +
27.5 25,5 2&3 24.3
± + ± +
17.5 25:5 35.1 35.4
298 TABLE II Haemodynamic effects o f noradrenaline microinjections into the nucleus tractus solitarius o f rats after local bilateral pretreatment with saline or 1)-(CH2)5- Tyr(Me)AVP
A unilateral microinjection of noradrenaline (20 nmol) was given 10 rain after bilateral microinjections of either saline or D-(CHz)5Tyr(Me)AVP (9 pmol each side) into the nucleus tractus solitarius. Values represent changes from mean arterial pressure just before the injections of noradrenaline, and are expressed as mean _+S.E.M. *P < 0.05 vs saline pre-treated group (Duncan's multiple-range test); '~P< 0.01; *P < 0.005; ~ not significant (one-way analysis of variance); n = number of rats. Time after noradrenaline injection (min)
A Mean arterialpressure (mm Hg)
2 5 1(I 15
- 3.6_+2 - 21+6.4 -32.3 _+7.5 -24.3+6.1
Saline/ noradrenaline* (n = 5)
D-(CH2)5-Tyr(Me)-AVP/ Saline/ noradrenaline* noradrenaline * (n = 6) (n = 5)
4.7+2 - 7.1 +2.8* -14.4 + 4.1" - 13_+3.7
aline was injected alone (Fig. 2). These rats also developed a mild bradycardia, but this did not reach statistical significance. Microinjections of n o r a d e n a l i n e 10 min after the local administration of saline in the same place decreased both blood pressure and heart rate (P < 0.005). This response was similar to that observed after local p r e t r e a t m e n t with either AVP or oxytocin (Table I).
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Noradrenaline
Fig. 3. Haemodynamic effects observed 2 and 10 min after microinjections of 10 pmol noradrenaline alone (SAL) or simultaneously with AVP, DDAVP or oxytocin (OXT) into the nucleus tractus solitarius of Sprague-Dawley rats. The peptides were used at a dose of 0.9 pmol. The number of rats is in brackets. *P < 0.05 (Duncan's multiple-range test).
- 74_+20.1 -100+20.1 -138 ___16.7 -132_+ 18.l
1)-(CH2)s-Tyr(Me)-AVP/ noradrenaline n (n = 6)
-43_+25.7 -45_+24.7 -60 + 24.3* -65_+30.5
Values of mean arterial pressure and heart rate 10 rain after bilateral injections of either saline or p(CH2)s-Tyr(Me)-AVP were not significantly different from basal values. A t this time, an unilateral injection of n o r a d r e n a l i n e was administered, and this resulted in a depressor response in both groups. H o w e v e r , in the V~ antagonist p r e t r e a t e d group, the m a x i m u m fall in blood pressure and heart rate was significantly smaller than that observed in the saline p r e t r e a t e d animals (Table II). A further experiment was p e r f o r m e d using a subthreshold dose of noradrenaline (10 pmol). A t this dose, the amine p r o d u c e d non-detectable effects on the cardiovascular p a r a m e t e r s tested. H o w e v e r , when this was administered simultaneously with AVP, an increase in both mean arterial pressure and heart rate was registered (Fig. 3). In contrast, simultaneous administration of noradrenaline (10 pmol) and either D D A V P or the other n e u r o h y p o p h y s e a l peptide, oxytocin, p r o d u c e d no significant changes in blood pressure and heart rate (Fig. 3). Basal values of mean arterial pressure and heart rate in vasopressin-deficient B r a t t l e b o r o rats were 83.8 + 4.7 mm Hg and 370 + 17 beats p e r min, respectively. In these animals, microinjections of saline or 0.9 pmol AVP into the NTS p r o d u c e d no appreciable changes in blood pressure and heart rate. A higher dose of AVP (9 pmol), however, elicited a m a r k e d rise in both cardiovascular p a r a m e t e r s (Table III). This response d e v e l o p e d rapidly and lasted m o r e than 20 min. In a further experiment, n o r a d r e n a l i n e (20 nmol)
299 TABLE III Haemodynamic effects o f A V P microinjections into the nucleus tractus solitarius o f vasopressin-deficient Brattleboro rats
Values represent changes from basal mean arterial pressure, and are expressed as mean ± S.E.M, *P < 0.05 v,. saline qDuncan's multiple-range test); n = number of rats. Time after
A Mean arterial pressure tram Hg)
injection (min)
Saline
4 V P O.9
AVP 9
Saline
A V P O. 9
(n = 51
pmol ¢n = 6)
pmoI (n = 5~
(n = 5J
pmol (n = oJ
umol ~n ~ 51
0.3 ___2.7 -3.3 + 1.2 -7.1 ___1.6 -7.4___1.9 -7.1 + 2.1
1.6 z 2.6 5.3 * 3.6 - 8.6 ~- 3.9 -10.0___5.3 - 9.4 ~- 5.4
8.3 x 3.110.1 +_2.7* 9.7 _+ 1.9' 11.1±2.2" 12.2 4--2 1"
_ ,1 z 2.7 -14 ± 4.5 -22 _~5.5 -26+7.6 -26 _ 7.6
-15 z 7.4 -16 ~ 4.6 -23 _~3.6 -25_+3.7 -23 _+5.4
24 -_- 18.0 ~,~~ 20.4* c~llz 2(/.1" 687-20* 65 _+ 18.6"
2 5 10 15 20
AHeart rate ¢beats/min i
was microinjected into the right NTS of both Brattleboro and parent strain L o n g - E v a n s rats. In L o n g - E -
1V P u
sponse was very similar to that observed after the ad-
vans rats, the amine significantly decreased (P <
ministration of n o r a d r e n a l i n e alone in the NTS of L o n g - E v a n s rats.
0.01) m e a n arterial pressure and heart rate (Fig. 4). This response was qualitatively similar to that ob-
DISCUSSION
served in S p r a g u e - D a w l e y rats. In Brattleboro rats. however, a pressor response (P < 0.005) was observed after the administration of n o r a d r e n a l i n e
There is evidence available that both catecholamines 19'2s and AVP 13"24 may play a role in the con-
(Fig. 4). This response developed rapidly, reaching maximal values within two min. and lasted about 10 min, after which blood pressure returned to basal values without further changes. At the same time. a rapid bradycardia (P < 0.02) also developed (Fig. 4). W h e n n o r a d r e n a l i n e and the subthreshold dose of AVP (0.9 pmol) were injected simulaneously, a decrease in blood pressure rather than a pressor effect was observed, and this was also accompanied by a rapidly developed bradvcardia (Fig. 4). This re-
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Fig. 4. Haemodynamic effects observed 2 and 10 min after microinjections of 20 nmol noradrenaline into the nucleus tractus solitarius of Long-Evans (LE, n = 6) or vasopressin-deficient Brattleboro (BRT, n = 5) rats. In some Brattleboro rats noradrenaline was microinjected simultaneously with a subthreshold dose (0.9 pmol1 of AVP (n = 5).
trol of cardiovascular homeostasis in the NTS. The innervation with vasopressin and oxytocin fibres from the hypothalamus, and the content of catecholaminergic cell bodies and terminals, renders the NTS a prime location for interactions between these putative neurotransmitters. In the present study, we present data suggesting that neurohypophyseal peptides work together with n o r a d r e n a l i n e in the control of blood pressure in this area of the brainstem. Our data show that simultaneous administration of AVP or oxytocin into the NTS of rats completely abolished the hypotensive response to noradrenaline, a transient pressor effect even being observed with AVP. These interactions occurred at doses of AVP and oxytocin which had n o cardiovascular effects when given alone. Local p r e t r e a t m e n t with these peptides did not modify the response to noradrenaline, indicating that the target neurones must be activated concurrently by both the amine and the peptides. In addition, coinjections of a subthreshold dose of both noradrenaline and AVP produced a pressor effect. It is noticeable that the nature o f the effect of AVP did not differ according to the dose of noradrenaline employed. In both cases co-injection of AVP and n o r a d r e n a l i n e yielded values of m e a n arterial pressure and heart rate higher than those observed when the amine was administered alone, no
300 matter whether it produced hypotension or not. The existence of receptors for AVP in the NTS has been reported from autoradiographic studies 2 and inferred from the fact that AVP specifically alters cardiovascular parameters when administered in this area 24. On the contrary, the existence of oxytocin receptors in the NTS has proved difficult to demonstrate, since specific binding sites in this area have not been detected 6'25, and this peptide does not alter cardiovascular function when administered alone into the NTS, even at a dose 10-fold higher than that used in this study 24. However, oxytocin has been found in presynaptic terminals in the NTS 26, and can be released from this area in a calcium-dependent manner 4. In addition, in spite of the lack of evidence for the existence of specific binding sites, oxytocin has a stimulatory action on neurones of the dorsal motor nucleus of the vagus 5, a region functionally and anatomically related to the NTS. Since it is unlikely that oxytocin acts on AVP receptors 12, a detailed study to characterize putative oxytocin receptors in this area is needed. In the periphery, AVP acts on at least two types of receptors 12. V 1 receptors, which activate phosphatidyl inositol turnover, are present in vascular smooth muscle and liver and mediate AVP-induced vasoconstriction and glycogenolysis. V 2 receptors, which activate adenylate cyclase, are found on the renal tubular cells and are responsible for the antidiuretic effects of this peptide. It is unknown whether VI and/or V~ receptors are present within the central nervous system, and we investigated the type of receptor responsible for our observed response by using DDAVP, a specific V2 agonist analog of AVP. Since DDAVP, unlike AVP, did not alter the response to noradrenaline, it suggests that the interaction between AVP and this amine is not due to activation of V, receptors. The possible involvement of V l receptors was also investigated by the administration of D-(CH2) 5Tyr(Me)-AVP, a specific V l antagonist 1~, which was found with no effects when given alone. Since co-injection of AVP abolished the hypotensive response to noradrenaline, it could be expected that administration of a vasopressin antagonist would potentiate or at least would not modify this response. On the contrary, our results suggest that D-(CH2)5-Tyr(Me )AVP has an agonistic effect, since it markedly re-
duced the hypotension and bradycardia induced by noradrenaline in the same way that AVP did, but with less potency. In a previous study 24. we have found that the pressor response elicited by microinjections of a higher dose of AVP (9 pmol) in the NTS was inhibited by local pretreatment with D-(CH2) 5Tyr(Me)-AVP. However, in a similar study, this compound has previously been found to produce a pressor effect on its own 13. Previous studies have suggested that central nervous system and pituitary vasopressin receptors are not clearly similar to the VI or V 2 receptors characterized in the periphery, and in fact they may represent a totally different type (V39)1"12. Our results now suggest that at the NTS also AVP receptors may have different characteristics so that I)-(CH2)5-Tyr(Me)-AVP behaves as a partial agonist as well as antagonist. If the interaction between noradrenaline and AVP has a functional significance, it might be expected that the haemodynamic responses to one of them would be altered in the absence of the other. This hypothesis was tested in vasopressin-deficient Brattleboro rats. In these animals, microinjections of AVP resulted in a pressor effect. We have previously characterized a similar response in S p r a g u e - D a w l e y rats 24, and our results now suggest that although Brattleboro rats cannot synthetise AVP, they do seem to have functional receptors for this peptide. Microinjections of noradrenaline into the NTS of these rats resulted in a pressor rather than a depressor effect. In the absence of endogenous AVP, the response to the amine was therefore clearly altered. The mechanism for this adaptation is unclear, although it is known, for example, that Brattleboro rats have an altered noradrenergic innervation of the hypothalamus from brainstem nuclei 1;. These changes in the innervation of brain nuclei could be very important in determining the nature of the response. Of remarkable interest is the fact that the altered response to noradrenaline in these animals was reversed by simultaneous administration of a subthreshold dose of AVP. In the light of their lack of endogenous AVP, this change in the haemodynamic response to noradrenaline is consistent with a physiological role for AVP in the NTS of normal animals. Interactions between a classical neurotransmitter and a peptide have been studied only to a limited extent, and only in the periphery has their possible
301 f u n c t i o n a l significance b e e n clearly d e s c r i b e d ~. A t
transmitters
the level of t h e spinal c o r d t h e r e is e v i d e n c e suggest-
target n e u r o n e s , w i t h o u t eliciting r e s p o n s e s o n t h e i r
r e c e i v e d s i m u l t a n e o u s l y on the s a m e
ing that t h y r o t r o p i n - r e l e a s i n g h o r m o n e and s e r o t o -
own.
nin i n t e r a c t in the m e d i a t i o n of sexual r e s p o n s e s 7. W e h a v e n o w s h o w n in the b r a i n s t e m that an i n t e r a c -
ACKNOWLEDGEMENTS
tion b e t w e e n an a m i n e and a p e p t i d e n e u r o t r a n s m i t ter results in m a r k e d c h a n g e s in c a r d i o v a s c u l a r func-
W e wish to t h a n k P r o f e s s o r M. M a n n i n g ( D e p a r t -
tion, e v e n at s u b t h r e s h o l d doses of b o t h c o m p o u n d s ,
ment
suggesting a r o l e for A V P in m o d i f y i n g the r e s p o n s e to the a c t i v a t i o n of n o r a d r e n e r g i c p a t h w a y s in the
O H ) for t h e gift of the v a s o p r e s s i n antagonist. M , V . was in r e c e i p t of a ' P l a n d e F o r m a c i 6 n d e P e r s o n a l In-
N T S . This strongly s u p p o r t s the c o n c e p t 3 that s o m e
v e s t i g a d o r ' F e l l o w s h i p , f r o m t h e Spanis h M i n i s t r y of
n e u r o t r a n s m i t t e r s m o d i f y the e f f e c t i v e n e s s of o t h e r
E d u c a t i o n and Science.
REFERENCES
propionic acid)] arginine-vasopressm, two highly potent antagonists of the vasopressor response to arginine-vasopressin. J. Meal Chem.. 23 (1980) 364-368. 12 Manning, M. and Sawyer. W.H.. Design and uses of selectwe agonistic and antagonistic analogs of the neuropeptides oxytocin and vasopressin, TINS. 711984)6-9. 13 Matsuguchi, H.. Sharabi, F.M.. Gordon. F.J.. Johnson. A.K. and Schmid. P.G., Blood pressure and heart rate responses to mxcroinjection of vasopressin into the nucleus tractus solitarius region of the rat. Neuropharmacology, 21 (1982) 687-693. 14 Palkovits. M. and Zaborszky. L.. Neuroanatomv of central cardiovascular control. Nucleus tractus solitarius: afferent and efferent connections in relation to the baroreceptor reflex arc. In A.P. Provoost and A.P. Shapiro (Eds.), Hypertension and Brain Mechanisms. Progress in Brain Research. Vol. 47. Elsevier. Amsterdam. 1977. pp. 9-34. 15 Pellegrino, L.L. Pellegrino. A.S. and Cushman, A.J.. A Stereotaxic Atlas of the Rat Brain. Plenum. New York, 1979. 16 Sawchenko. P.E. and Swanson. L.W., lmmunohistochemical identification of neurons in the paraventricular nucleus of the hypothalamus that project to the medulla or to the spinal cord in the rat. J. Comp. Neurol.. 205 (1982) 260-272 17 Scholer. J. and Sladek. J.R.. Supraoptic nucleus of the Brattleboro rat has an altered afferent noradrenergic input. Science. 214 (1981) 347-349. 18 Sladek. J.R. and Sladek. C.D.. Anatomical reciprocity between magnocellular peptides and noradrenaline in putatwe cardiovascular pathways. In B A. Cross and G. Leng (Eds.), The Neurohypophysis: Structure, Function and Control. Progress in Brain Research. Vol. 60, Elsevier. Amsterdam, 1983, pp. 437-443. 19 Snyder. D.W., Nathan, M.A. and Reis. D.J., Chronic lability of arterial pressure produced by selective destruction of the catecholamine innervation of the tractus solitarii m the rat. Circ. Res.. 43 (1978) 262-27l. 20 Sofroniew. M.V. and Schrell,V., Evidence for a direct projection from oxytocin and vasopressin neurons in the hypothalamic paraventricular nucleus to the medulla oblongata: immunohistochemical visualization of both the horseradish peroxidase transported and the peptide produced by the same neurons, Neurosci. Lett., 22 (1981) 211-217. 21 Sumal. K.K., Blessing, W.W.. John. T.H.. Reis, D.J, and
1 Antoni, F.A., Novel ligand specificity of pituitary vasopressin receptors in the rat, Neuroendocrinology, 39 (1984) 186-188. 2 Biegon, A., Terlou, M., Voorhuis, T.D. and De Kloet, E.R., Arginine-vasopressin binding sites in the rat brain: a quantitative autoradiographic study, Neurosci. Lett., 44 (1984) 229-234. 3 Bloom, F.E., The functional significance of neurotransmitter diversity, Am. J. Physiol., 246 (1984) C184-C194. 4 Buijs, R.M. and Van Heerikhuize, J.J., Vasopressin and oxytocin release in the brain - a synaptic event, Brain Research, 252 (1982) 71-76. 5 Charpak, S., Armstrong, W.E., Muhlethaler, M. and Dreifuss, J.J., Stimulatory action of oxytocin on neurones of the dorsal motor nucleus of the vagus nerve, Brain Research, 300 (1984) 83-89. 6 DeKloet, E.R., Rotteveel, F., Voorhuis, T.D. and Terlou, M., Topography of binding sites for neurohypophyseal hormones in rat brain, Eur. J. Pharmacol., 110 (1985) 113-119. 7 Hansen, S., Svenson, L., H6kfelt, T. and Everitt, B.J., 5hydroxytryptamine-thyrotropin releasing hormone interactions in the spinal cord: effects on parameters of sexual behaviour in the male rat, Neurosci. Lett., 42 (1983) 299-304. 8 H6kfett, T., Everitt, B., Holets, V.R., Meister, B., Melander, T., SchaUing, M., Staines, W. and Lundberg, J.M., Coexistence of peptides and other active molecules in neurons: diversity of chemical signaling potential. In L.L. Iversen and E. Goodman (Eds.), Fast and Slow Chemical Signaling in the Nervous System, Oxford University Press, Oxford, 1986, pp. 205-231. 9 Kalia, M., Woodward, D.J., Smith, W.K. and Fuxe, K., Rat medulla oblongata. IV. Topographical distribution of catecholaminergic neurons with quantitative three-dimensional computer reconstruction, J. Comp. Neurol., 233 (1985) 350-364. 10 Koda, L.Y. and Bloom, F.E., Distribution of catecholamine-containing cell bodies and blood vessels in the rat nucleus tractus solitarius, Brain Research, 289 (1983) 71-78. 11 Kruszynski, M., Lammek, B., Manning, M., Seto, J., Haldar, J. and Sawyer, W.H., [1-(fl-Mercapto-fl,fl-cyclopentamethylenepropionic acid), 2-(0-methyl) tyrosine] argininevasopressin and [1-(/3-mercapto-fl,fl-cyclopentamethylene-
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302
22
23
24
25
Pickel, V.M., Synaptic interaction of vagal afferents and catecholaminergic neurons in the rat nucleus tractus solitarius, Brain Research, 277 (1983) 31-40. Takahashi, Y., Satoh, K., Sakumoto, I., Tohyama, M. and Shimizu, N., A major source of catecbolamine terminals in the nucleus tractus solitarii, Brain Research, 172 (1979) 372-377. Tanaka, M., DeKloet, E.R., DeWied, D. and Versteeg, D.H.G., ArginineS-vasopressin affects catecholamine metabolism in specific brain nuclei, Life Sci., 20 (1977) 1799-1808. Vallejo, M., Carter, D.A. and Lightman, S.L., Haemodynamic effects of arginine-vasopressin microinjections into the nucleus tractus solitarius: a comparative study of vasopressin, a selective vasopressin receptor agonist and antagonist, and oxytocin, Neurosci. Lett., 52 (1984) 247-252. Van Leeuwen, F.W., Van Heerikhuize, J., Van der Meu-
len, G. and Wolters, P., Light microscopic autoradiographic localization of [3H]oxytocin binding sites in the rat brain, pituitary and mammary gland, Brain Research, 359 (1985) 320-325. 26 Voorn, P. and Buijs, R.M., An immuno-electronmicroscopical study comparing vasopressin, oxytocin, substance P and enkephalin-containing nerve terminals in the nucleus of the solitary tract of the rat, Brain Research, 270 (1983) 169-173. 27 White, J.D., Krause, J.E. and McKelvy, J.F., In vivo biosynthesis and transport of oxytocin, vasopressin and neurophysins to posterior pituitary and nucleus of the solitary tract, J. Neurosci., 4 (1984) 1262-1270. 28 Zandberg, P., DeJong, W. and DeWied, D., Effect ofcatecholamine-receptor stimulating agents on blood pressure after local application in the nucleus tractus solitarii of the medulla oblongata, Eur. J. Pharmacol., 55 (1979) 43-56.
295
BRE 12920
Evidence for a functional relationship between noradrenaline and neurohypophyseal peptides in the brainstem of rats Mario Vallejo and Stafford L. Lightman Medical Unit, Charing Cross and Westminster Medical School, London ( U. K.) (Accepted 3 March 1987) Key words: Neurohypophyseal peptide; Noradrenaline; Peptide/amine interaction; Nucleus tractus soiitarius: Blood pressure; Brattleboro rat
The possibility of a functional relationship between noradrenaline and neurohypophyseal peptides in the control of cardiovascular function in the nucleus tractus solitarius of rats has been investigated. The hypotensive response to microinjections of noradrenaline (20 nmolI was abolished by simultaneous (but not prior) microinjections of [ArgS]vasopressin (AVP) and oxytocin at doses (0.9 pmoB which alone had no effects on cardiovascular parameters. AVP plus noradrenaline actually resulted in a transient pressor effect. Simul. taneous administration of [deamino-D-ArgS]vasopressm. a selective agonist of AVP V z receptors, did not modify the effect of noradrenaline, whereas the specific V 1 antagonist D-(CH2)5-Tyr(Me)-AVP partially decreased its intensity. When subthreshold doses of both AVP (0.9 pmol) and noradrenaline (10 pmol) were administered simultaneously, a pressor response was observed. In vasopressin-deficient Brattleboro rats. mlcroinjections of 0.9 pmol AVP had no effects, but a marked pressor response was observed after the administration of a higher dose (9 pmol). In parent strain Long-Evans rats. noradrenaline (20 nmol) also produced a hypotensive re~ sponse, but in Brattleboro rats microinjections of this amine elicited a marked pressor effect. In these rats. simultaneous administra~ tion of a subthreshold dose of AVP (0.9 pmol) reversed this response in such a way that a fall in blood pressure, similar totha~ observed in Long-Evans rats after injection of noradrenaline alone, was registered. These results provide evidence for a functional interaction between noradrenafine and neurohypophyseal peptides in the control of cardiovascular function in the brainstem.
INTRODUCTION The nucleus tractus solitarius (NTS) within the dorsomedial m e d u l l a contains a rich p o p u l a t i o n of noradrenergic neurons of the A 2 group 9'1°. which are a m a j o r source of catecholaminergic terminals in this area 22. This nucleus also receives a p r o j e c t i o n of neur o h y p o p h y s e a l p e p t i d e - c o n t a i n i n g fibres which originate from cell bodies in the h y p o t h a l a m i c parvocellular paraventricular nucleus 16'2°. It is now known that these peptides are synthesized in the h y p o t h a l a m u s and carried by axonal t r a n s p o r t to the NTS 27. In this area, neurophysin-containing varicosities have b e e n described in juxtaposition with n o r a d r e n a l i n e perikarya and dendrites is. Cardiovascular afferent fibres from pressure r e c e p t o r s in the thorax travel in the IX and X nerves to the NTS 14. where some vagal fibres
project to n o r a d r e n e r g i c n e u r o n e s of the A 2 group 2~ [ArgS]vasopressm (AVP) has been shown to alter noradrenaline t u r n o v e r in the NTS 23. a n d b o t h AVP and n o r a d r e n a l i n e alter cardiovascular function when injected into this nucleus 13'24"2s. The coexistence of amine and p e p t i d e neurotransmitters within the s a m e n e u r o n e s or their presence in pathways projecting to the same target cells strongly suggests the existence of interactions b e t w e e n them. Functional interactions have now been clearly described in the p e r i p h e r y s. In the brain, however, a relationship b e t w e e n amines and p e p t i d e s has b e e n reported in vitro, but physiologically functional interactions have proved difficult to d e m o n s t r a t e s . A l though the anatomical distribution of n e u r o h y p o p h y seal p e p t i d e s and amine n e u r o n s in the NTS suggests a possible interaction in the control of cardiovascular
Correspondence: M. Vallejo. Present address: MRC Molecular Neurobiology Unit. University of Cambridge Medical School. Hills Road, Cambridge CB2 2QH, U.K 0006-8993/87/$03.50 © 1987 Elsevier Science Publishers B.V. ~Biomedical Division ~
296 function, no studies have hitherto addressed this possibility. The present experiments were designed to look for evidence of any functional relationship between noradrenaline and neurohypophyseal peptides in the NTS of rats. MATERIALS AND METHODS
Male Sprague-Dawley rats (250-350 g) were anaesthetized with urethane (1.3 g/kg i.p.). The left common carotid artery was cannulated with polythene tubing and connected via a Statham P23 ID transducer to a Gould Thermal chart recorder for continuous measurement of blood pressure and heart rate. The animals were placed in a Kopf stereotaxic frame with the head flexed downwards to an angle of 45 °. After a midline incision through the skin, the dorsal neck muscles were retracted and the atlantooccipital membrane was incised in order to expose the dorsal surface of the brainstem. After 20-30 rain, an injection (0.2/.tl delivered over 10 s) was administered into the right NTS, using pulled glass micropipettes ( - 5 0 ~m in diameter) connected by plastic tubing to a 10-~1 Hamilton syringe driven by a microinfusion pump. The injections were placed at the level of the caudal tip of the area postrema, 0.3 mm lateral to the midline and 0.6 mm below the dorsal surface of the brainstem. Each rat received only one injection, unless otherwise indicated. In order to confirm correct placement, histological examination of frozen sections of the brains was carried out in rats injected with safranine into the same position at the end of the experiments. Sections were cut at 25 ~m, stained with thionine and compared to the atlas of Pellegrino et al. ~5, revealing that the injectate was distributed within the NTS area, with minimal diffusion into the adjacent dorsal motor nucleus of the vagus. Rats were injected with vehicle (0.9% saline), ( - ) noradrenaline bitartrate (Sigma, U.K.), [ArgS]vaso pressin (Cambridge Research Biochemicals, U.K.), oxytocin (Cambridge Research Biochemicals, U.K.), [deamino-D-ArgS]vasopressin (DDAVP) (Ferring, Malmo, Sweden) or D-(CHjs-Tyr(Me)AVP, a specific antagonist of V 1 receptors 11. All the peptides were administered at a dose of 0.9 pmol, except the V 1 antagonist, which was given at a dose of 9 pmol. Noradrenaline was administered at a dose of
either 10 pmol or 20 nmol. Some animals treated with saline, AVP or oxytocin received a further injection of noradrenaline (20 nmol) into the same position 10 min later. Simultaneous administration of noradrenaline and peptides was also performed. Noradrenaline was disolved in a solution of saline containing the appropriate amount of AVP, oxytocin, DDAVP or D(CH2)5-Tyr(Me)-AVP. The dual injection was administered unilaterally so that 0.9 pmol of each peptide (or 9 pmol in the case of the V~ antagonist) was coinjected with either 10 pmol or 20 nmol noradrenaline. One further group of rats received bilateral injections of either saline or D-(CH2)5-Tyr(Me)-AVP (9 pmol each side), and 10 min later a unilateral injection of noradrenaline (20 nmol). In another study, male AVP-deficient Brattleboro and parent strain Long-Evans rats (220-300 g) were subjected to the general procedure described above, and treated with single microinjections of noradrenaline (20 nmol) in the right NTS. In addition, some Brattleboro rats were given microinjections of either saline or AVP (0.9 or 9 pmol), Simultaneous administration of AVP (0.9 pmol) and noradrenaline (20 nmol) was also performed in these animals. All Brattleboro rats were infused throughout the experi-
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Fig. i. H a e m o d y n a m i c effects of m i c r o i n j e c t i o n s of 20 n m o l n o r a d r e n a l i n e ([], n = 6), 0.9 p m o l A V P ( A , n = 5) or b o t h
substances simultaneously (©, n = 6) into the nucleus tractus solitarius of Sprague-Dawley rats.
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Fig. 2. Haemodynamic effects observed 2 and 10 min after microinjections of 20 nmol noradrenaline alone (SALI or simultaneously with DDAVP, AVP, oxytocin (OXT), or the V 1 antagonist D-(CH2)5-Tyr(Me)-AVP (ANT) into the nucleus tractus solitarius of Sprague-Dawley rats. All the peptides were administered at a dose of 0.9 pmol, except the vasopressin antagonist which was given at a dose of 9 pmol. The number of rats is in parentheses; *P < 0.05 (Duncan's multiple-range test).
ments with 0.9% saline via a cannula inserted in the jugular vein, at a rate of 6 ml/h, in o r d e r to avoid dehydration. L o n g - E v a n s rats were also cannulated but not infused. The statistical analysis of cardiovascular changes within groups was c a r r i e d out using t h e one-way analysis of variance, unless otherwise indicated. Duncan's multiple-range test was used for multiple comparisons b e t w e e n groups. In this case statistical significance was accepted for P < 0.05.
Basal values of mean arterial pressure and heart rate in S p r a g u e - D a w l e y rats were 93.3 _+ 5.1 m m H g and 403.8 + 15 beats p e r min. respectively. N o significant alterations of cardiovascular p a r a m e t e r s were observed after microinjections of saline, AVP, oxytocin, D D A V P or o-(CH2)5-Tyr(Me)-AVP. A significant decrease ( P < 0.005) in both b l o o d pressure and h e a r t rate was h o w e v e r o b s e r v e d in response to microinjections of 20 nmol n o r a d r e n a l i n e (Fig. l). This hypotensive effect d e v e l o p e d rapidly and was maximal within 10 min. lasting for m o r e than 20 min. W h e n AVP and n o r a d r e n a l i n e were injected simultaneously, a transient but significant mcrease (P < 0.02. Student's t-test) in b l o o d pressure was observed (Fig. 1). This p r e s s o r response only lasted 5 min, following which m e a n arterial p r e s s u r e r e t u r n e d to basal values without further changes. A small decrease in heart rate was seen, but this did not achieve statistical significance (Fig. 1). Simultaneous administration of oxytocin a n d n o r a d r e n a l i n e p r o d u c e d no appreciable changes in cardiovascular p a r a m e t e r s (Fig. 2). A m a r k e d decrease in both mean arterial pressure and h e a r t rate ( P < 0.005) was o b s e r v e d after simultaneous administration of D D A V P and noradrenaline, and this r e s p o n s e was not significantly different from that o b s e r v e d when the a m i n e was injected alone (Fig. 2). A significant decrease (P < 0.005) in m e a n arterial pressure was also o b s e r v e d after simultaneous administration of noradrenatine and D-(CH2)5-Tyr(Me)-AVP. H o w e v e r , the maxim u m fall, o b s e r v e d 10 min after the injection, was significantly less than that o b s e r v e d when noradren-
TABLE I Haemodynamic effects of noradrenaline microinjections into the nucleus tractus solitarius of rats, after local pre-treatment with saline, AVP or oxytocin
Noradrenaline (20 nmol) was administered 10 rain after microinjection of saline, AVP (0.9 pmol) or oxytocin (0.9 pmol) into the same place. Values represent changes from mean arterial pressure just before the injection of noradrenaline, and are expressed as mean + S.E.M. of 6 experiments. *P < 0.05, one-way analysis of variance; tp < 0.02, ~P < 0~005. Time after noradrenaline
A Mean arterial pressure (mm Fig)
d Heart rate (beats/rain)
injection (min)
Saline/ noradrenaline*
AVP/ noradrenaline*
Oxytocin/ noradrenaline ~
Saline/ noradrenaline ~
AVP/ noradrenalme*
Oxytocin/ noradrenaline
2 5 10 15
-4.5±3.2 -13±3.7 -22±3.2 -~±3.6
- 5±2.4 -15.5±3.6 -~.5±4.5 -18.6±4.2
- 3.6±5,6 -17.5±2.5 -I8.6±4.4 -13.6±4.4
-38±6.5 -~±9.9 -7±11;1 -70±10.3
-65 -78 -88 -91
-51 "61 -70 -66
+ + ± +
27.5 25,5 2&3 24.3
± + ± +
17.5 25:5 35.1 35.4
298 TABLE II Haemodynamic effects o f noradrenaline microinjections into the nucleus tractus solitarius o f rats after local bilateral pretreatment with saline or 1)-(CH2)5- Tyr(Me)AVP
A unilateral microinjection of noradrenaline (20 nmol) was given 10 rain after bilateral microinjections of either saline or D-(CHz)5Tyr(Me)AVP (9 pmol each side) into the nucleus tractus solitarius. Values represent changes from mean arterial pressure just before the injections of noradrenaline, and are expressed as mean _+S.E.M. *P < 0.05 vs saline pre-treated group (Duncan's multiple-range test); '~P< 0.01; *P < 0.005; ~ not significant (one-way analysis of variance); n = number of rats. Time after noradrenaline injection (min)
A Mean arterialpressure (mm Hg)
2 5 1(I 15
- 3.6_+2 - 21+6.4 -32.3 _+7.5 -24.3+6.1
Saline/ noradrenaline* (n = 5)
D-(CH2)5-Tyr(Me)-AVP/ Saline/ noradrenaline* noradrenaline * (n = 6) (n = 5)
4.7+2 - 7.1 +2.8* -14.4 + 4.1" - 13_+3.7
aline was injected alone (Fig. 2). These rats also developed a mild bradycardia, but this did not reach statistical significance. Microinjections of n o r a d e n a l i n e 10 min after the local administration of saline in the same place decreased both blood pressure and heart rate (P < 0.005). This response was similar to that observed after local p r e t r e a t m e n t with either AVP or oxytocin (Table I).
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Fig. 3. Haemodynamic effects observed 2 and 10 min after microinjections of 10 pmol noradrenaline alone (SAL) or simultaneously with AVP, DDAVP or oxytocin (OXT) into the nucleus tractus solitarius of Sprague-Dawley rats. The peptides were used at a dose of 0.9 pmol. The number of rats is in brackets. *P < 0.05 (Duncan's multiple-range test).
- 74_+20.1 -100+20.1 -138 ___16.7 -132_+ 18.l
1)-(CH2)s-Tyr(Me)-AVP/ noradrenaline n (n = 6)
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Values of mean arterial pressure and heart rate 10 rain after bilateral injections of either saline or p(CH2)s-Tyr(Me)-AVP were not significantly different from basal values. A t this time, an unilateral injection of n o r a d r e n a l i n e was administered, and this resulted in a depressor response in both groups. H o w e v e r , in the V~ antagonist p r e t r e a t e d group, the m a x i m u m fall in blood pressure and heart rate was significantly smaller than that observed in the saline p r e t r e a t e d animals (Table II). A further experiment was p e r f o r m e d using a subthreshold dose of noradrenaline (10 pmol). A t this dose, the amine p r o d u c e d non-detectable effects on the cardiovascular p a r a m e t e r s tested. H o w e v e r , when this was administered simultaneously with AVP, an increase in both mean arterial pressure and heart rate was registered (Fig. 3). In contrast, simultaneous administration of noradrenaline (10 pmol) and either D D A V P or the other n e u r o h y p o p h y s e a l peptide, oxytocin, p r o d u c e d no significant changes in blood pressure and heart rate (Fig. 3). Basal values of mean arterial pressure and heart rate in vasopressin-deficient B r a t t l e b o r o rats were 83.8 + 4.7 mm Hg and 370 + 17 beats p e r min, respectively. In these animals, microinjections of saline or 0.9 pmol AVP into the NTS p r o d u c e d no appreciable changes in blood pressure and heart rate. A higher dose of AVP (9 pmol), however, elicited a m a r k e d rise in both cardiovascular p a r a m e t e r s (Table III). This response d e v e l o p e d rapidly and lasted m o r e than 20 min. In a further experiment, n o r a d r e n a l i n e (20 nmol)
299 TABLE III Haemodynamic effects o f A V P microinjections into the nucleus tractus solitarius o f vasopressin-deficient Brattleboro rats
Values represent changes from basal mean arterial pressure, and are expressed as mean ± S.E.M, *P < 0.05 v,. saline qDuncan's multiple-range test); n = number of rats. Time after
A Mean arterial pressure tram Hg)
injection (min)
Saline
4 V P O.9
AVP 9
Saline
A V P O. 9
(n = 51
pmol ¢n = 6)
pmoI (n = 5~
(n = 5J
pmol (n = oJ
umol ~n ~ 51
0.3 ___2.7 -3.3 + 1.2 -7.1 ___1.6 -7.4___1.9 -7.1 + 2.1
1.6 z 2.6 5.3 * 3.6 - 8.6 ~- 3.9 -10.0___5.3 - 9.4 ~- 5.4
8.3 x 3.110.1 +_2.7* 9.7 _+ 1.9' 11.1±2.2" 12.2 4--2 1"
_ ,1 z 2.7 -14 ± 4.5 -22 _~5.5 -26+7.6 -26 _ 7.6
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2 5 10 15 20
AHeart rate ¢beats/min i
was microinjected into the right NTS of both Brattleboro and parent strain L o n g - E v a n s rats. In L o n g - E -
1V P u
sponse was very similar to that observed after the ad-
vans rats, the amine significantly decreased (P <
ministration of n o r a d r e n a l i n e alone in the NTS of L o n g - E v a n s rats.
0.01) m e a n arterial pressure and heart rate (Fig. 4). This response was qualitatively similar to that ob-
DISCUSSION
served in S p r a g u e - D a w l e y rats. In Brattleboro rats. however, a pressor response (P < 0.005) was observed after the administration of n o r a d r e n a l i n e
There is evidence available that both catecholamines 19'2s and AVP 13"24 may play a role in the con-
(Fig. 4). This response developed rapidly, reaching maximal values within two min. and lasted about 10 min, after which blood pressure returned to basal values without further changes. At the same time. a rapid bradycardia (P < 0.02) also developed (Fig. 4). W h e n n o r a d r e n a l i n e and the subthreshold dose of AVP (0.9 pmol) were injected simulaneously, a decrease in blood pressure rather than a pressor effect was observed, and this was also accompanied by a rapidly developed bradvcardia (Fig. 4). This re-
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Fig. 4. Haemodynamic effects observed 2 and 10 min after microinjections of 20 nmol noradrenaline into the nucleus tractus solitarius of Long-Evans (LE, n = 6) or vasopressin-deficient Brattleboro (BRT, n = 5) rats. In some Brattleboro rats noradrenaline was microinjected simultaneously with a subthreshold dose (0.9 pmol1 of AVP (n = 5).
trol of cardiovascular homeostasis in the NTS. The innervation with vasopressin and oxytocin fibres from the hypothalamus, and the content of catecholaminergic cell bodies and terminals, renders the NTS a prime location for interactions between these putative neurotransmitters. In the present study, we present data suggesting that neurohypophyseal peptides work together with n o r a d r e n a l i n e in the control of blood pressure in this area of the brainstem. Our data show that simultaneous administration of AVP or oxytocin into the NTS of rats completely abolished the hypotensive response to noradrenaline, a transient pressor effect even being observed with AVP. These interactions occurred at doses of AVP and oxytocin which had n o cardiovascular effects when given alone. Local p r e t r e a t m e n t with these peptides did not modify the response to noradrenaline, indicating that the target neurones must be activated concurrently by both the amine and the peptides. In addition, coinjections of a subthreshold dose of both noradrenaline and AVP produced a pressor effect. It is noticeable that the nature o f the effect of AVP did not differ according to the dose of noradrenaline employed. In both cases co-injection of AVP and n o r a d r e n a l i n e yielded values of m e a n arterial pressure and heart rate higher than those observed when the amine was administered alone, no
300 matter whether it produced hypotension or not. The existence of receptors for AVP in the NTS has been reported from autoradiographic studies 2 and inferred from the fact that AVP specifically alters cardiovascular parameters when administered in this area 24. On the contrary, the existence of oxytocin receptors in the NTS has proved difficult to demonstrate, since specific binding sites in this area have not been detected 6'25, and this peptide does not alter cardiovascular function when administered alone into the NTS, even at a dose 10-fold higher than that used in this study 24. However, oxytocin has been found in presynaptic terminals in the NTS 26, and can be released from this area in a calcium-dependent manner 4. In addition, in spite of the lack of evidence for the existence of specific binding sites, oxytocin has a stimulatory action on neurones of the dorsal motor nucleus of the vagus 5, a region functionally and anatomically related to the NTS. Since it is unlikely that oxytocin acts on AVP receptors 12, a detailed study to characterize putative oxytocin receptors in this area is needed. In the periphery, AVP acts on at least two types of receptors 12. V 1 receptors, which activate phosphatidyl inositol turnover, are present in vascular smooth muscle and liver and mediate AVP-induced vasoconstriction and glycogenolysis. V 2 receptors, which activate adenylate cyclase, are found on the renal tubular cells and are responsible for the antidiuretic effects of this peptide. It is unknown whether VI and/or V~ receptors are present within the central nervous system, and we investigated the type of receptor responsible for our observed response by using DDAVP, a specific V2 agonist analog of AVP. Since DDAVP, unlike AVP, did not alter the response to noradrenaline, it suggests that the interaction between AVP and this amine is not due to activation of V, receptors. The possible involvement of V l receptors was also investigated by the administration of D-(CH2) 5Tyr(Me)-AVP, a specific V l antagonist 1~, which was found with no effects when given alone. Since co-injection of AVP abolished the hypotensive response to noradrenaline, it could be expected that administration of a vasopressin antagonist would potentiate or at least would not modify this response. On the contrary, our results suggest that D-(CH2)5-Tyr(Me )AVP has an agonistic effect, since it markedly re-
duced the hypotension and bradycardia induced by noradrenaline in the same way that AVP did, but with less potency. In a previous study 24. we have found that the pressor response elicited by microinjections of a higher dose of AVP (9 pmol) in the NTS was inhibited by local pretreatment with D-(CH2) 5Tyr(Me)-AVP. However, in a similar study, this compound has previously been found to produce a pressor effect on its own 13. Previous studies have suggested that central nervous system and pituitary vasopressin receptors are not clearly similar to the VI or V 2 receptors characterized in the periphery, and in fact they may represent a totally different type (V39)1"12. Our results now suggest that at the NTS also AVP receptors may have different characteristics so that I)-(CH2)5-Tyr(Me)-AVP behaves as a partial agonist as well as antagonist. If the interaction between noradrenaline and AVP has a functional significance, it might be expected that the haemodynamic responses to one of them would be altered in the absence of the other. This hypothesis was tested in vasopressin-deficient Brattleboro rats. In these animals, microinjections of AVP resulted in a pressor effect. We have previously characterized a similar response in S p r a g u e - D a w l e y rats 24, and our results now suggest that although Brattleboro rats cannot synthetise AVP, they do seem to have functional receptors for this peptide. Microinjections of noradrenaline into the NTS of these rats resulted in a pressor rather than a depressor effect. In the absence of endogenous AVP, the response to the amine was therefore clearly altered. The mechanism for this adaptation is unclear, although it is known, for example, that Brattleboro rats have an altered noradrenergic innervation of the hypothalamus from brainstem nuclei 1;. These changes in the innervation of brain nuclei could be very important in determining the nature of the response. Of remarkable interest is the fact that the altered response to noradrenaline in these animals was reversed by simultaneous administration of a subthreshold dose of AVP. In the light of their lack of endogenous AVP, this change in the haemodynamic response to noradrenaline is consistent with a physiological role for AVP in the NTS of normal animals. Interactions between a classical neurotransmitter and a peptide have been studied only to a limited extent, and only in the periphery has their possible
301 f u n c t i o n a l significance b e e n clearly d e s c r i b e d ~. A t
transmitters
the level of t h e spinal c o r d t h e r e is e v i d e n c e suggest-
target n e u r o n e s , w i t h o u t eliciting r e s p o n s e s o n t h e i r
r e c e i v e d s i m u l t a n e o u s l y on the s a m e
ing that t h y r o t r o p i n - r e l e a s i n g h o r m o n e and s e r o t o -
own.
nin i n t e r a c t in the m e d i a t i o n of sexual r e s p o n s e s 7. W e h a v e n o w s h o w n in the b r a i n s t e m that an i n t e r a c -
ACKNOWLEDGEMENTS
tion b e t w e e n an a m i n e and a p e p t i d e n e u r o t r a n s m i t ter results in m a r k e d c h a n g e s in c a r d i o v a s c u l a r func-
W e wish to t h a n k P r o f e s s o r M. M a n n i n g ( D e p a r t -
tion, e v e n at s u b t h r e s h o l d doses of b o t h c o m p o u n d s ,
ment
suggesting a r o l e for A V P in m o d i f y i n g the r e s p o n s e to the a c t i v a t i o n of n o r a d r e n e r g i c p a t h w a y s in the
O H ) for t h e gift of the v a s o p r e s s i n antagonist. M , V . was in r e c e i p t of a ' P l a n d e F o r m a c i 6 n d e P e r s o n a l In-
N T S . This strongly s u p p o r t s the c o n c e p t 3 that s o m e
v e s t i g a d o r ' F e l l o w s h i p , f r o m t h e Spanis h M i n i s t r y of
n e u r o t r a n s m i t t e r s m o d i f y the e f f e c t i v e n e s s of o t h e r
E d u c a t i o n and Science.
REFERENCES
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