Enkephalins, substance P, bradykinin and angiotensin II: Differential sites of action on the hypothalamo-neurohypophysial system

Brain Research, 220 (1981) 107-119

107

Elsevier/North-Holland Biomedical Press

E N K E P H A L I N S , SUBSTANCE P, B R A D Y K I N I N A N D A N G I O T E N S I N II: D I F F E R E N T I A L SITES OF A C T I O N ON T H E H Y P O T H A L A M O N E U R O H Y P O P H Y S I A L SYSTEM

A. J. BAERTSCHI, H. H. ZINGG* and J. J. DREIFUSS (Accepted November 6th, 1980) Departments of Physiology and of Animal Biology, University of Geneva, 1211 Geneva 4 (Switzerland) Key words: angiotensinII--bradykinin--enkephalin--hypothalamo-neurohypophysialsystem--

substance P

SUMMARY To test the hypothesis that enkephalins, substance P, bradykinin and angiotensin II could act as neurohumoral modulators of hypothalamic function, these peptides (0.01-20 /zg) were injected into the general circulation of anesthetized rats, and changes in hypothalamo-neurohypophysial activity were determined by continuously monitoring the amplitude of antidromic compound action potentials (CAP) in the hypothalamo-hypophysial tract. A decrease of CAP amplitude was taken to indicate an increase of orthodromic impulse traffic. All peptides elicited a CAP decrease. On a molar basis when injected i.v., the enkephalin analog F K 33-824 was the most effective substance, followed by substance P, Leu-enkephalin and angiotensin II. Enkephalins and substance P injected through the internal carotid artery, were 2-5 times more effective than when injected i.v., whereas bradykinin was most effective when it reached the brain through a vertebral route. Angiotensin II produced the same CAP decrease irrespective of the route of administration and, in contradistinction to the other peptides, its effect was not abolished by stalk section. Tachyphylaxis and reversibility with naloxone was observed only for the enkephalins. The data suggest that sites of action are the hypothalamus for enkephalins and substance P, the neurohypophysis for angiotensin II, and the hindbrain for bradykinin.

* Present address: Department of Medicine, Royal Victoria Hospital and McGill University, Montreal l13A 1A1, Canada. 0006-8993/81/0000-0000/$02.50© Elsevier/North-Holland Biomedical Press

108 INTRODUCTION

Enkephalin, substance P, bradykinin and angiotensin 11 have been found in the mammalian brainT,lZ,ls, 23 as well as in the general circulation11,12,20, 23. These peptides share two features, i.e. (a) their role as neuromodulators or neurotransmitters, and (b) their importance in the neuroendocrine control of the circulation. Substance p34 and bradykinin 25 are potent vasodilators; angiotensin II is a vasoconstricting and thirst-producing substance23; endorphins have been implicated in hypovolemic shock 15. Both bradykinin 26 and enkephalins 6, when injected intravenously, can elicit the release of antidiuretic hormone (ADH), but their sites of action are not clearly delineated. To test whether these peptides when carried by the blood stream may play a role in neuroendocrine mechanisms, we studied the effects and the probable sites of action of substance P, enkephalins, angiotensin II and bradykinin on the electrical activity of the hypothalamo-neurohypophysial system. Preliminary results have been presented previously2,L METHODS

Experiments were conducted on urethane anesthetized (0.13 g/100 g body weight) rats (250-350 g) of either sex, some of which were lactating. The hypothalamoneurohypophysial activity was recorded as described previously 4. Briefly, the hypothalamus was exposed by a transpharyngeal approach 14, and a bipolar stimulating electrode was positioned on the hypophysial stalk, near its junction with the hypophysis. Compound antidromic action potentials (CAP) were recorded within the hypothalamo-hypophysial tract, 1.2-1.4 mm rostral to the stimulation electrode, in response to constant current (400-1000 /~A) bipolar impulses (0.4-1.0 msec pulse width) applied through the stimulation electrode at a steady frequency of 1.0-2.0 Hz. The CAP amplitude has been shown to decrease whenever the orthodromic impulse traffic of the hypothalamo-hypophysial axons increases, and this decrease is correlated with neurohypophysial hormone release 4. Substance P, angiotensin II and bradykinin were purchased from Beckman, while the enkephalin analog F K 33-824 (Tyr-D-Ala-Gly-MePhe-Met-[O]-ol)and Leu-enkephalin were kindly donated by Sandoz Ltd., Basel. These peptides were dissolved in saline and were bolus injected into various segments of the general circulation: (a) intravenously; (b) into the common carotid artery towards the brain to reach mainly the hypothalamus and the forebrain; (c) into the common carotid artery towards the heart in order to reach initially the hindbrain. In some cases, substance P or Leu-enkephalin was also injected into the descending aorta or into the internal or external carotid arteries (see Fig. 8). Similar methods have been applied previously3,s to determine presumptive sites of drug action, such as the forebrain, the bulbo-pontine region or areas lying outside the CNS. To check if the drugs could directly act on the hypothalamo-neurohypophysial axons, a V-shaped cut was performed on the basal hypothalamus, the bottom of the V lying halfway between the origin of the portal vessels and the optic chiasma and the sides of the V along the walls of the third ventricle.

109 This lesion interrupted the hypothalamo-neurohypophysial axons without injury to portal vessels. The peak amplitude of the CAP was monitored on-line by means of a voltage holder and continuously recorded on chart together with aortic pressure (through a common carotid artery catheter) and, in the lactating rats, milk ejection pressure. The signals including a synchronisation pulse from the electrical stimulator, were also stored on magnetic tape for further analysis. The changes in CAP amplitude in response to peptide injection were usually expressed as percentages of preinjection values; the latter ranged from 4 to 7 mV (see Fig. 9). The percentage decrease of CAP amplitude from all animals injected with a given substance were pooled to yield means and standard errors. Differences in means were analyzed for statistical significance by Student's t-test. RESULTS

Bradykinin When injected at doses of 1-10/~g into the jugular vein or into the common carotid artery, bradykinin elicited a dose-dependent decrease of CAP amplitude. A typical chart recording (Fig. 1A) shows that arterial blood pressure decreased concomitantly with CAP amplitude. However, hypotension was not the cause of the CAP decrease, since inhalation of amyl nitrite had no detectable effect on CAP amplitude while producing an even larger decrease of arterial pressure (Fig. 1B). In doses up to 5/~g, bradykinin was significantly (P < 0.05) more effective in decreasing CAP amplitude when the intracarotid injection was directed toward the heart (i.c.h.) than when it was directed toward the brain (i.c.b.); i.c.h, injection was also more effective than intravenous injection (P < 0.05). CAP responses to 10 #g bradykinin injected by either route were not significantly different (P :> 0.4), nor were those to i.v. or i.c.b, injections at all doses used (P > 0.3) (Fig. 2A). The area under the CAP decreased more rapidly and to a greater degree when bradykinin was injected i.c.h, rather than i.v. (Fig. 2B), suggesting that the principal site of action of bradykinin was located within a CNS territory irrigated by the vertebral artery,

Angiotensin 11 Unlike bradykinin, angiotensin II in doses up to 10 #g elicited a small but prolonged decrease of CAP amplitude (Fig. 3A, left panel). This decrease was unchanged when the hypothalamo-hypophysial tract had been transected at the level of the median eminence, prior to angiotensin injection (Fig. 3B, left panel). The CAP decrease under the latter condition could not have been due to incomplete tract lesioning, since the sharp CAP decrease observed in response to vaginal distension (Fig. 3A, right panel) was abolished by the lesion (Fig. 3B, right panel). In a group of 4 animals, the CAP response to angiotensin II following the transection of the fiber tract was preserved (Fig. 4B), whereas the responses to either nicotine tartrate (100 #g i.v.) or vaginal distension were abolished. Neither could the CAP decrease have been consecutive to electrode movement with respect to the center of the fiber tract, since

110

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motions of the recording electrode by ~ 30 #m did not restore the CAP amplitude to control level (Fig. 3B, left panel). The effect of angiotensin II was dose-dependent and independent of the route of administration (Fig. 4A). Thus angiotensin II, in all likelihood, acted on the tract either by exerting a direct effect on to the axons or by affecting them indirectly via an effect on the portal circulation. With respect to the latter hypothesis, it should be noted that hypertension produced by injection ofnoradrenaline was not accompanied by any decrease of the CAP amplitude.

Enkephalins and substance P The Met-enkephalin analog F K 33-824 (I yg, i.v.) elicited within seconds a marked decrease of CAP amplitude; in lactating rats this response was followed by an increase of intramammary pressure (Fig. 5A, B). The effect was reversed by naloxone (Fig. 5C, D). Substance P, at doses of 1-10 yg, produced an equally large and prolonged CAP decrease. Dose-response curves (Fig. 6) showed that the minimum effective dose for obtaining a detectable CAP decrease was 0.1 #g for the enkephalin analog, 1 #g for substance P and 2.5 yg for Leu-enkephalin. The effect of 1 yg enkephalin analog was significantly larger than that of 2.5/zg substance P (P < 0.05) and 10 #g Leu-enkephalin (P < 0.05). The usual hypotension elicited by the enkephalins and substance P never exceeded that evoked by bradykinin or by amyl nitrite. When the animals had a low mean blood pressure, substance P provoked an unexpected rise in arterial blood pressure, but the changes of CAP amplitude were similar to those observed in normotensive animals.

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Fig. 2. Effects of increasing concentrations of bradykinin. A: decrease of CAP amplitude (measured at time of maximal decrease, see Fig. 1A) following injections of 1-10/~g bradykinin either intravenously (O), or into the common carotid artery, with the injection directed towards the brain (O) and towards the heart (&). Results are means from 3-4 animals. For statistical significance, see text. B" changes of CAP area (measured between baseline and CAP by integration) to 5 :tg bradykinin injected at time 0 (arrow) in one animal. Symbols as in A. CAP amplitude is sensitive to orthodromic impulse traffic and spread of individual action potentials, while CAP area reflects mainly orthodromic impulse traffic. In most instances, decrease of CAP amplitude was proportional to decrease of CAP area, as shown previously4. R e p e a t e d a p p l i c a t i o n o f the e n k e p h a l i n a n a l o g at 8 min intervals lead to t a c h y p h y l a x i s a n d the response recovered only p a r t i a l l y at 64 min intervals (Fig. 7A). I n contrast, we o b s e r v e d n o c h a n g e in the a m p l i t u d e o f the response to r e p e a t e d injections o f substance P (10 /~g i.v.) (Fig. 7B). R e p e a t e d a p p l i c a t i o n o f Leue n k e p h a l i n at 8 min intervals l e a d to a 30-40 ~ loss o f response ( n o t shown). T h e injection o f substance P (2-5 # g ) into the c o m m o n c a r o t i d a r t e r y t o w a r d s the b r a i n was 2-3 times m o r e effective t h a n i n t r a v e n o u s injection o f the s a m e doses

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(Fig. 8); intra-aortic or i n t r a c a r o t i d injections directed t o w a r d s the heart were least effective. The partial tachyphylaxis observed with the enkephalins m a d e it m o r e difficult to study their site o f action. Nevertheless, injections o f L e u - e n k e p h a l i n into the internal c a r o t i d artery were estimated to be 2-5 times m o r e effective ( P < 0.05) than external carotid or intravenous injections. A typical experiment is shown in Fig. 9. As was the case for b r a d y k i n i n a n d nicotine, transection o f the h y p o t h a l a m o - n e u r o h y p o p h y s i a l tract abolished the effects o f substance P a n d o f e n k e p h a l i n s on the C A P amplitude.

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Fig. 4. Effects of angiotensin II. A: decrease of CAP amplitude to injections of 1-10/~g angiotensin II applied intravenously (O), into the common carotid towards the brain (O), and towards the heart (A) (one animal). B: changes of CAP amplitude following transection of the hypothalamo-neurohypophysial tract, to angiotensin II (A II), to nicotine tartrate and to vaginal distension (VD). Results are means 4- S.E.M. of 4 animals, expressed relative to the CAP decrease observed before the lesion.

DISCUSSION The results suggest that increasing the plasma level of enkephalins, substance P and bradykinin causes an acceleration of orthodromic neural activity in the hypothalamo-neurohypophysial tract and, consequently, may enhance the rate of secretion of vasopressin and/or oxytocin from the posterior pituitary gland 4. The enkephalin analog F K 33-824, probably owing to its resistance to enzymatic degradation 27, was the most potent substance tested. It was followed, in order of potency, by substance P, bradykinin and Leu-enkephalin. Angiotensin I1, even at high concentrations, had little effect on the impulse traffic in the hypothalamo-neurohypophysial tract. The small CAP decrease observed was apparently due to a direct action of angiotensin II on the neurohypophysial axons or on their ionic environment, since

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117 general circulation can elicit the release of ADH. Our data show that this action is independent of the hypotensive effects exerted by these peptides and results from an action on the central nervous system. One site of action of enkephalin appears to be a region of the brain irrigated by the internal carotid arteries, and this may include the hypothalamus. As noted previously3, the principal site of action for bradykinin appears to be a region irrigated by the vertebral arteries. Angiotensin injected into cerebrospinal fluid and into the diencephalon causes the release of vasopressin3°,a6 and excites magnocellular endocrine neurons 1, possibly by a direct action on the hypothalamic perikarya2Z,2L The role of plasma angiotensin II in neurohypophysial hormone release remains controversial (see ref. 23). In conscious dogs, intravenous infusions of 10 ng/kg/min angiotensin II elicited the release of vasopressin and of ACTH 24. In anesthetized dogs, however, intracarotid infusions of angiotensin II had no effect on basal ADH release, but potentiated the release of ADH in response to intravenous hypertonic NaC131. The data presented in this report suggest that plasma angiotensin II acts on hypothalamo-neurohypophysial axons, a finding in accord with claims that isolated posterior pituitary lobes kept in vitro release ADH in response to nanomolar concentrations of angiotensin I116,17, 35. Other neuromodulators or neurotransmitters, including y-aminobutyric acid37, enkephalin1°,19 and catecholamines21, are also believed to act on hypothalamoneurohypophysial axons. To our knowledge there are only two reports2, 9 in the literature on the effects of substance P on neurohypophysial function. Our data suggest that substance P acts on a central site irrigated by the internal carotid artery to cause posterior pituitary hormone secretion. An effect was observed for lower doses than were needed with the naturally occurring enkephalin. Nevertheless, the physiological importance of substance P remains to be established. The plasma concentrations of substance P reached after injection can be estimated to be well above the highest physiological levels measured in the rat 2°. This probably also holds true for enkephalin11, angiotensin II 33 and even bradykinin12, although plasma bradykinin can increase to very high levels under pathological conditions. Even higher doses than those used in this study were applied by other investigators and were found to induce pain 32 or analgesia33. It remains to be explored if these peptides can cross the blood-brain barrier or if their action on the CNS is exerted indirectly, for example via the circumventricular organs. Since the hypothalamus is particularly rich in endogenous enkephalinTM and substance pT, an alternative possibility to be considered is that these peptides may reach high concentrations in the hypothalamic microcirculation, and may thereby modulate posterior pituitary hormone release. ACKNOWLEDGEMENTS This study was supported in part by Grants 3.248.77 and 3.469.79 from the Swiss National Science Foundation. We thank Ms. D. Machard for technical assistance.

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119 27 Roehmer, D., Buescher, H. H., Hill, R. C., Hess, J., Bauer, W., Cardinaux, F., Closse, A., Hauser, D. and Huguenin, R.A., Synthetic enkephalin analogue with prolonged parenteral and oral analgesic activity, Nature (Lond.), 268 (1977) 547-549. 28 Rossier, J., Battenberg, E., Pittman, Q., Bayon, A., Koda, L., Miller, R., Guillemin, R. and Bloom, F., Hypothalamic enkephalin neurones may regulate the neurohypophysis, Nature (Lond.), 277 (1979) 653-655. 29 Sakai, K. K., Marks, B. H., George, J. and Koestler, A., Specific angiotensin II receptors in organ-cultured canine supraoptic nucleus cells, Life Sci., 14 (1974) 1337-1344. 30 Simonnet, G., Rodriguez, F., Fumoux, F., Czernichow, P. and Vincent, J. D., Vasopressin release and drinking induced by intracranial injection of angiotensin II in monkey, Amer. J. Physiol., 237 (1979) R20-R25. 31 Shimizu, K., Share, L. and Claybaugh, J. R., Potentiation by angiotensin II of the vasopressin response to an increasing plasma osmolality, Endocrinology, 93 (1973) 42-50. 32 Taira, N., Nakayama, K. and I-Iashimato, K., Vocalization response to puppies to intra-arterial administration of bradykinin and other algesic agents, and mode of action of blocking agents, Tohoku J. exp. Med., 96 (1968) 365-377. 33 Urca, G., Frenk, H., Liebeskind, J. C. and Taylor, A. N., Morphine and enkephalin: analgesic and epileptic properties, Science, 197 (1977) 83-86. 34 Von Euler, U. S., and Gaddum, J. H., An unidentified depressor substance in certain tissue extracts, J. Physiol. (Lond.), 72 (1931) 74-87. 35 Wagner, H., H~iberle, M. and Maier, V., The effect of angiotensin II on arginine vasopressin release from the rat hypothalamus and hypophysis in vitro, Proc. Int. Union Physiol. Sci., 13 (1977) 796 (abstract). 36 Yamamoto, M., Share, L. and Shade, R. E., Effect of ventriculocisternal perfusion with angiotensin II and indomethacin on the plasma vasopressin concentration, Neuroendocrinology, 25 (1978) 166-173. 37 Zingg, H. H., Baertschi, A. J. and Dreifuss, J. J., Action of ),-aminobutyricacid on hypothalamoneurohypophysial axons, Brain Research, 171 (1979) 453-459.