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Effect of intracerebroventricular 5,6-dihydroxytryptamine on blood pressure of spontaneously hypertensive rats
European Journal of Pharmacology, 36 (1976) 431--437 © North-Holland Publishing Company, Amsterdam -- Printed in The Netherlands
431
EFFECT OF INTRACEREBROVENTRICULAR 5,6-DIHYDROXYTRYPTAMINE ON BLOOD PRESSURE OF SPONTANEOUSLY HYPERTENSIVE RATS ROBIN E. BUCKINGHAM, THOMAS C. HAMILTON and DAVID ROBSON Pharmacology Department, Roche Products Limited, Welwyn Garden City, Hertfordshire, England
Received 19 August 1975, revised MS received 14 October 1975, accepted 29 December 1975
R.E. BUCKINGHAM, T.C. HAMILTON and D. ROBSON, Effect of intracerebroventricular 5,6-dihydroxytryptamine on blood pressure of spontaneously hypertensive rats, European J. Pharmacol. 36 (1976) 431--437. The effects of intracerebroventricular injections of 5,6-DHT on the development and maintenance of hypertension in spontaneously hypertensive rats has been investigated. 5,6-DHT, injected into 6 week old rats, retarded the development of hypertension for at least 6 weeks; this effect was not accompanied by inhibition of the pressor response produced by stimulation of the total peripheral sympathetic outflow. 5,6-DHT, injected into 14--15 week old rats with established hypertension, produced a short-lived fall in blood pressure. These findings suggest that central 5-HT neurones are involved in the development of hypertension in spontaneously hypertensive rats. Spontaneously hypertensive rats Intracerebroventricular
5,6-Dihydroxytryptamine
1. I n t r o d u c t i o n T h e role o f brain 5 - h y d r o x y t r y p t a m i n e (5HT) as a f u n c t i o n a l t r a n s m i t t e r in the central nervous s y s t e m is s u p p o r t e d b y e x p e r i m e n t a l evidence (Chase and M u r p h y , 1 9 7 3 ) , and whilst 5-HT secreting n e u r o n e s have b e e n identified in v a s o m o t o r regions o f t h e brain (DahlstrSm and F u x e , 1965; F u x e et al., 1 9 6 8 ) t h e i r role in the regulation o f b l o o d pressure has n o t b e e n established. I n t r a c e r e b r o v e n t r i c u l a r (i.c.v.) administrat i o n o f 5 , 6 - d i h y d r o x y t r y p t a m i n e (5,6-DHT) causes a selective d e s t r u c t i o n o f central 5-HT c o n t a i n i n g nerve terminals ( B a u m g a r t e n et al., 1 9 7 2 c ) with a r e s u l t a n t d e p l e t i o n o f brain 5-HT levels ( B a u m g a r t e n et al., 1971, 1 9 7 2 a ) and r e d u c t i o n o f n e u r o n a l 5-HT u p t a k e (BjSrklund et al., 1 9 7 3 ) . Intracisternally administered 5,6-DHT prevents the d e v e l o p m e n t o f n e u r o g e n i c , b u t n o t renal, h y p e r t e n s i o n in rabbits (Wing and Chalmers, 1 9 7 4 ) , b u t does n o t m o d i f y t h e d e v e l o p m e n t o f DOCA/saline
Blood pressure
h y p e r t e n s i o n in rats (Myers et al., 1974}. In t h e present study, 5,6-DHT administered i.c.v. has b e e n used t o evaluate t h e role o f central 5-HT secreting n e u r o n e s in the d e v e l o p m e n t and m a i n t e n a n c e o f raised b l o o d pressure in s p o n t a n e o u s l y h y p e r t e n s i v e rats.
2. Materials and methods 2.1. A n i m a l s
Male s p o n t a n e o u s l y h y p e r t e n s i v e rats (SHrats) (Japanese strain; O k a m o t o and Aoki, 1963) were used f o r these studies. 2.2. D i r e c t recording o f conscious SH-rats
blood pressure in
U n d e r e t h e r anaesthesia, a p o l y t h e n e cannula was i m p l a n t e d in the a b d o m i n a l a o r t a o f rats, using a m o d i f i c a t i o n o f the m e t h o d described by Weeks and J o n e s (1960). T h e rats
432
were allowed 1--2 days to recover before use. Blood pressure was recorded in the conscious unrestrained animal using a Statham P23 Db pressure transducer; heart rate was counted from the record after increasing the chart speed.
2.3. Stimulation of sympathetic outflow in pithed SH-rats SH-rats were set up in pairs (one control and one treated) for stimulation of the entire sympathetic outflow by the method described by Gillespie and Muir (1967). Each rat received i.v. atropine, 1 mg/kg, and d-tubocurarine, 2 mg/kg, after pithing. Preparations were electrically stimulated for periods of 20 sec every 8 min, by monophasic, square-wave pulses of 0.5 msec duration, at supramaximal voltage, over the frequency range 0.25--8 Hz. Changes in diastolic blood pressure were recorded from the indwelling aortic cannula using a Statham P23 Db pressure transducer.
2.4. Drug treatment with i.c.v. 5,6-DHT 2.4.1. SH-rats, 6 weeks old (95--105 g), received an i.c.v, injection of 5,6-DHT, 25 pg, or vehicle, 10 pl of 0.9% w/v saline containing 0.1 mg/ml ascorbic acid, in a manner similar to that described for mice by Haley and McCormick (1957). A 0.5 ml Hamilton syringe with a No. 25 needle was used, together with a No. 20 needle as guide tube. Injections were made to a depth of 3.5 mm in animals lightly anaesthetized with ether. The accuracy of the method was verified in other young rats by location of injected dye in the lateral ventricle. After injection animals were housed in groups of 8 until they attained the required age. At age 8, 10 and 12 weeks, the blood pressure and heart rate (see 2.2.) and body weight of groups of treated and control rats were measured and, on the same day, the animals were pithed and stimulation of the sympathetic outflow was performed (see 2.3.).
R.E. BUCKINGHAM ET AL.
Additionally the blood pressure and heart rate of 6 week old rats were measured by the direct recording method (see 2.2. ). 2.4.2. SH-rats, 14--15 weeks old (260-310 g) received i.c.v, injections via a cannula implanted 3 or 4 days previously according to the method of Hayden et al. (1966). Blood pressure and heart rate were recorded (see 2.2.) in the same group of animals at 24 hr intervals on 5 days {days 0--4). 5,6-DHT, 50 pg i.c.v., or vehicle, 20 pl of 0.9% w/v saline containing 0.1 mg/ml ascorbic acid, was injected immediately after zero time recordings on day 0. In a further group of animals blood pressure and heart rate were recorded at hourly intervals for 4 hr after injection.
2. 5. Drugs used 5,6-Dihydroxytryptamine creatinine sulphate (kindly supplied by Dr. Langemann, F. Hoffman--La Roche, Basle) was dissolved in 0.9% w/v saline containing 0.1 mg/ml ascorbic acid. Doses are expressed as the base. Results are expressed as means + standard error of the mean. Data were analysed using Student's ttest and significance of difference taken at p ~< 0.05.
3. Results
3.1. Effect of pretreatment with 5,6-DHT (25 pg i.c.v.) on the development o f hypertension 2, 4 and 6 weeks after treatment of 6 week old rats with 5,6-DHT, the blood pressure of treated rats was significantly lower than that of age-matched control rats treated with vehicle. At each age the blood pressure of control animals was significantly higher (14--20 mm Hg) (p <: 0.01 at 8 weeks; p < 0.001 at 10 and 12 weeks) than that of treated rats (fig. 1A). Whilst the blood pressure of control rats significantly increased by 18 mm Hg from age 6--8 weeks and by 42 mm Hg from age 6--12 weeks, the blood pressure of 8 week old treated rats was not significantly different from
5,6-DIHYDROXYTRYPTAMINE AND BLOOD PRESSURE
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Fig. 1. Change in (A) mean arterial pressure (BP mm Hg,), (B) heart rate (HR beats/min), (C) body weight (Wt g), with age (weeks), of spontaneously hypertensive rats treated at 6 weeks age with 5,6-DHT~ 25 pg i.c.v. (~ --) or with 10 ~l vehicle (© ©). Groups of 9--12 animals were used; vertical bars extend to S.E.M.
t h a t o f 6 w e e k old rats, a n d at 12 w e e k s age t h e b l o o d pressure of t h e t r e a t e d g r o u p was 22 m m Hg higher t h a n t h a t o f 6 w e e k old rats. T h e h e a r t r a t e o f 8, 10 and 12 w e e k old c o n t r o l and t r e a t e d rats was significantly lower (p < 0.01 at each age) t h a n t h a t o f 6 w e e k old rats; at each age the h e a r t r a t e o f t r e a t e d rats was n o t significantly d i f f e r e n t f r o m t h a t o f a g e - m a t c h e d c o n t r o l a n i m a l s (fig. 1B). A t age 8, 10 a n d 12 weeks t h e b o d y w e i g h t o f t r e a t e d rats was significantly less ( 1 6 - - 3 5 g) (p < 0.01 at each age) t h a n t h a t o f a g e - m a t c h e d c o n t r o l animals. T h e b o d y weights o f 12 w e e k old c o n t r o l and t r e a t e d rats were r e s p e c t i v e l y 156 a n d 140 g greater t h a n t h o s e of 6 w e e k old rats (fig. 1C). T h e d a t a in figs. 1 A and 1C s h o w t h a t f o r t h e s a m e level o f b l o o d pressure in c o n t r o l and t r e a t e d rats, t h e t r e a t e d rats are n o t o n l y o l d e r t h a n c o n t r o l rats b u t also have higher b o d y weight. F o r e x a m p l e , for a b l o o d pressure level o f 120 m m Hg in b o t h groups, t h e b o d y w e i g h t o f c o n t r o l rats ( 1 0 8 g) is 30 g less t h a n t h a t o f t r e a t e d rats; similarly at 142 m m Hg t h e b o d y weight o f c o n t r o l rats ( 2 0 4 g) is 46 g less t h a n t h a t o f t r e a t e d rats.
3.2. Effect o f pretreatment with 5,6-DHT (25 lag i.c.v.) on neuronally induced pressor responses in pithed rats In a g e - m a t c h e d t r e a t e d a n d c o n t r o l rats, t h e f r e q u e n c y - d e p e n d e n t increases in diastolic
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Fig: 2. Relationship between increase in diastolic blood pressure (A BP mm Hg) and frequency of stimulation (Hz) of the spinal cord of pithed rats (age 8 weeks, [circles] and age 12 weeks [triangles] ) in animals pretreated at 6 weeks age with 5,6-DHT, 25 /2g i.c.v. ( e - - - - - 4 ) and (A. . . . . ~), or with 10 #l vehicle i.c.v. (o ©) and (~/x). Groups of 10 animals were used; vertical bars extend to S.E.M.
434
R.E. B U C K I N G H A M E T AL.
blood pressure produced by stimulation of the entire sympathetic outflow in pithed rats were not significantly different. Fig. 2 shows the relationship with frequency of the pressor responses evoked by neuronal stimulation in treated and control rats aged 8 and 12 weeks; similar results were obtained using 10 week old rats. Fig. 2 also shows that, other than stimulation at 0.25 and 0.5 Hz, the pressor responses to neuronal stimulation in 12 week old control rats were significantly higher (p < 0.001) than those evoked in 8 week'old rats; a similar significant difference existed for pressor responses in 10 and 12 week old rats.
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3.3. Effect of pretreatment with 5,6-DHT (50 pg i.c.v.) on the blood pressure of SH-rats with established hypertension 5,6-DHT significantly reduced (p < 0.001) the blood pressure of 14--15 week old SH-rats for at least 4 hr after injection; heart rate was significantly increased (p < 0.01) in treated animals 2--4 hr after injection (fig. 3). In a further experiment when measurements were made at 24 hourly intervals for 4 days after injection (fig. 4) the blood pressure and heart rate of treated animals did not differ from those of control rats.
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Fig. 3. T i m e course ( h r ) o f c h a n g e in (A) m e a n arterial b l o o d pressure (A BP m m Hg) a n d (B) h e a r t rate (A HR b e a t s / m i n ) o f 1 4 - - 1 5 w e e k old s p o n t a n e o u s l y h y p e r t e n s i v e rats following 5,6-DHT, 50 pg i.c.v. (~ e ) or 20 #l vehicle i.c.v. (o o). T h e initial m e a n arterial pressure a n d h e a r t r a t e o f each g r o u p were, respectively, for t r e a t e d r a t s 171 + 3 m m Hg a n d 381 + 8 b e a t s / m i n and, for u n t r e a t e d rats 170 +- 3 m m Hg a n d 388 -+ 10 b e a t s / m i n . G r o u p s o f 15 a n i m a l s were used; vertical bars e x t e n d t o S.E.M.
Fig. 4. T i m e course (days) o f (A) m e a n arterial pressure (BP m m Hg) a n d (B) h e a r t r a t e ( H R b e a t s / m i n ) of 1 4 - - 1 5 week old s p o n t a n e o u s l y h y p e r t e n s i v e rats t r e a t e d o n d a y 0 with 5,6-DHT, 50 pg i.c.v. (-, ) (n = 9) or 20 pl vehicle i.c.v. (o o) (n = 11). G r o u p s o f 9 - - 1 1 animals were used; vertical bars e x t e n d t o S.E.M.
5,6-DIHYDROXYTRYPTAMINE AND BLOOD PRESSURE 4. Discussion In this investigation, 5,6-DHT administered i.c.v., has been shown to retard, but not prevent, the development of hypertension in SHrats (Japanese strain) for at least 6 weeks, and to produce a short-lived fall in blood pressure in SH-rats with established hypertension. The doses used in this study have been shown by Baumgarten et al. (1971) to produce a marked and long-lasting reduction in brain 5-HT levels in the rat. Baumgarten et al. (1972c) showed that these effects of 5,6-DHT were the result of selective destruction of indoleamine containing nerve terminals. The retardation of the development of hypertension in our experiments suggests that 5-HT secreting neurones are involved in the central regulation of blood pressure in SH-rats; our findings would be consolidated by data showing the effect of 5,6-DHT in depleting 5-HT levels in the central nervous system of SH-rats. However, as Chalmers (1975) points out, the value of such studies is limited. Earlier studies showed that 6-hydroxydopamine (6-OHDA) administered i.c.v, to rats, produces a widepread destruction of the nerve terminals of central noradrenergic neurones (Uretsky and Iversen, 1970). In other studies 6-OHDA administered i.c.v, has been shown to inhibit the development of DOCA/saline, renal and spontaneous hypertension in the rat (Finch et al., 1972; Haeusler et al., 1972). In the rabbit 6-OHDA administered intracisternally, has been shown to inhibit the development of neurogenic and renal hypertension (Chalmers and Reid, 1972; Lewis et al., 1973). In recent studies Wing and Chaimers (1974) observed that intracisternal administration of 5,6-DHT produced a fall in blood pressure in normal and neurogenically hypertensive rabbits, and prevented the development of neurogenic, but not renal, hypertension in this species. In contrast, the development and maintenance of DOCA/saline hypertension in the rat were unaffected by intracisternally administered 5,6-DHT (Myers et al., 1974). These
435
observations, together with the results of our own studies, tend to suggest that unlike 6OHDA, 5,6-DHT demonstrates some degree of selectivity in its effect on the development of neurogenic hypertension in the rabbit and spontaneous hypertension in the rat. Wing and Chalmers (1974) suggested that the selective destruction of 5-HT containing bulbospinal nerves by 5,6-DHT in the rabbit interferes with the baroreceptor reflex arc and hence the development of neurogenic hypertension. It is possible that the retardation of the development of spontaneous hypertension in rats by 5,6-DHT which we have observed may be the result of an analogous mechanism. The failure of 5,6-DHT to reverse the raised blood pressure in SH-rats with established hypertension, suggests that 5-HT secreting netlrones may be involved in initiating the hypertension in young SH-rats, b u t may not play a significant role in maintaining the elevated pressure. A similar role has been ascribed to central noradrenergic neurones by Haeusler et al. (1972). This being so, there exists the possibility of a dual mechanism of initiation of hypertension in SH-rats. Such a dual control of peripheral sympathetic nerves and arterial blood pressure by bulbospinal nerves in the rabbit has been suggested by Chalmers (1975). It seems unlikely that an effect on brain catecholamines could contribute to the observed effects of 5,6-DHT in retarding the development of hypertension in SH-rats. Baumgarten et al. (1971) found that a rapid reduction in whole brain catecholamine levels followed the i.c.v, administration of 75 pg 5,6-DHT to normotensive rats. Normal catecholamine levels were, however, restored within 2--4 days of the injection. Furthermore the failure of 5,6-DHT to modify the increase in neuronally mediated pressor responses that occurred with age in SH-rats suggests that neither inhibition of peripheral sympathetic functional capacity nor the reduction in b o d y weight gain contributed to the impaired development of hypertension in these rats.
436
The acute fall in blood pressure elicited by i.c.v. 5,6-DHT in 14--15 week old SH-rats may be due to the initial release of endogenous brain noradrenaline and/or 5-HT (Baumgarten et al., 1971) or to the direct stimulant effect of 5,6-DHT on 5-HT receptors (Baumgarten et al., 1972b). There is a growing body of evidence to support a relationship between central 5-HT receptor stimulation and a fall in blood pressure. Bhargava and Tangri {1959) observed that i.c.v. 5-HT produces a marked fall in blood pressure in anaesthetised cats. Baum and Shropshire (1975) have recently demonstrated that i.v. 5-hydroxytryptophan (5-HTP), a precursor of 5-HT, given after peripheral decarboxylase inhibition, produces a progressive reduction in blood pressure and heart rate in anaesthetised cats. This effect is associated with a dose-dependent reduction in spontaneous electrical activity of cardiac, renal and splanchnic nerves. In contrast Henning and Rubenson (1971) showed that, in conscious rats, the hypotensive response to systematically administered 5-HTP is largely prevented by inhibition of peripheral decarboxylase. The acute antihypertensive response to i.c.v. 5,6-DHT in our experiments is unlikely to be peripherally mediated since i.v. 5,6DHT produces a pressor response in pithed rats (Baumgarten et al., 1972b).
Acknowledgement The authors are grateful to Mr. D.T. Burden for his technical assistance.
References Baum, T. and A.T. Shropshire, 1975, Inhibition of efferent sympathetic nerve activity by 5-hydroxytryptophan and centrally administered 5-hydroxytryptamine, Neuropharmacol. 14, 227. Baumgarten, H.G., A. BjSrklund, L. Lachenmayer, A. Nobin and U. Stenevi, 1971, Long-lasting selective depletion of brain serotonin by 5,6-dihydroxytryptamine, Acta Physiol. Scand., Suppl. 373. Baumgarten, H.G., K.D. Evetts, R.B. Holman, L.L.
R.E. BUCKINGHAM ET AL. Iversen, M. Vogt and G. Wilson, 1972a, Effects of 5,6-dihydroxytryptamine on monoaminergic neurones in the central nervous system of the rat, J. Neurochem., 19, 1587. Baumgarten, H.G., M. GSthert, H.G. Schlossberger and P. Tuchinda, 1972b, Mechanism of pressor effect of 5,6-dihydroxytryptamine in pithed rats, Naunyn-Schmiedeb. Arch. Pharmacol. 274, 375. Baumgarten, H.G., L. Lachenmayer and H.G. Schlossberger, 1972c, Evidence for a degeneration of indoleamine containing nerve terminals in rat brain induced by 5,6-dihydroxytryptamine, Z. Zellforsch. 125, 553. Bhargava, K.P. and K.K. Tangri, 1959, The central vasomotor effects of 5-hydroxytryptamine, Brit. J. Pharmacol. Chemoterap. 14, 411. BjSrklund, A., A. Nobin and U. Stenevi, 1973, Effects of 5,6-dihydroxytryptamine on nerve terminal serotonin and serotonin uptake in the rat brain, Brain Res. 53, 117. Chalmers, J.P., 1975, Brain amines and models of experimental hypertension, Circulation Res. 36, 469. Chalmers, J.P. and J.L. Reid, 1972, Participation of central noradrenergic neurones in arterial baroceptor reflexes in the rabbit. A study with intra-cisternally administered 6-hydroxydopamine, Circulation Res. 31,789. Chase, T.N. and D.L. Murphy, 1973, Serotonin and central nervous system function, Ann. Rev. Pharmacol. 13,181. DahlstrSm, A. and K. Fuxe, 1965, Evidence for the existence of monoamine-containing neurons in the central nervous system. I. Demonstration of monoamines in the cell bodies of brain stem neurones, Acta Physiol. Scand. 62, Suppl. 232. Finch, L., G. Haeusler and H. Thoenen, 1972, Failure to induce experimental hypertension in rats after intraventricular injection of 6-hydroxydopamine, Brit. J. Pharmacol. 44, 356. Fuxe, K., T. HSkfelt and U. Ungerstedt, 1968, Localisation of indolealkylamines in CNS, Advan. Pharmacol. 6A, 235. Gillespie, J.S. and T.C. Muir, 1967, A method of stimulating the complete sympathetic outflow from the spinal cord to blood vessels in the pithed rat, Brit. J. Pharmacol. Chemotherap. 30, 78. Haeusler, G., L. Finch and H. Thoenen, 1972, Central adrenergic neurones and initiation and development of experimental hypertension, Experientia 28, 1200. Haley, T.J. and W.G. McCormick, 1957, Pharmacological effects produced by intracerebral injection of drugs in the conscious mouse, Brit. J. Pharmacol. Chemotherap. 12, 12. Hayden, J.F., L.R. Johnson and R.P. Maickel, 1966, Construction and implantation of a permanent
5,6-DIHYDROXYTRYPTAMINE AND BLOOD PRESSURE cannula for making injections into the lateral ventricle of rat brain, Life Sci. 5, 1509. Henning, M. and A. Rubenson, 1971, Effects of 5-hydroxytryptophan on arterial blood pressure, body temperature and tissue monoamines in the rat, Acta Pharmacol. Toxicol. 29, 145. Lewis, P.J., J.L. Reid, J.P. Chalmers and C.T. Doilery, 1973, Importance of central catecholaminergic neurons in the development of renal hypertension, Clin. Sci. 45, 115. Myers, M.G., J.L. Reid and P.J. Lewis, 1974, The effect of central serotonin depletion on DOCA/saline hypertension "'l the rat, Cardiovascular Res. 8, 806.
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Okamoto, K. and K. Aoki, 1963, Development of a strain of spontaneously hypertensive rats, Jap. Circulation J. 27,282. Uretsky, N.J. and L.L. Ivecsen, 1970, Effects of 6-hydroxydopamine on catecholamine containing neurones in the rat brain, J. Neurochem. 17,269. Weeks, J.R. and J.A. Jones, 1960, Routine measurement of arterial pressure in unanaesthetised rats, Proc. Soc. Exptl. Biol. Med. 104, 646. Wing, L.M.H. and J.P. Chalmers, 1974, Participation of central serotonergic neurones in the control of the circulation of the unanaesthetised rabbit, Circulation Res. 35, 504.
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EFFECT OF INTRACEREBROVENTRICULAR 5,6-DIHYDROXYTRYPTAMINE ON BLOOD PRESSURE OF SPONTANEOUSLY HYPERTENSIVE RATS ROBIN E. BUCKINGHAM, THOMAS C. HAMILTON and DAVID ROBSON Pharmacology Department, Roche Products Limited, Welwyn Garden City, Hertfordshire, England
Received 19 August 1975, revised MS received 14 October 1975, accepted 29 December 1975
R.E. BUCKINGHAM, T.C. HAMILTON and D. ROBSON, Effect of intracerebroventricular 5,6-dihydroxytryptamine on blood pressure of spontaneously hypertensive rats, European J. Pharmacol. 36 (1976) 431--437. The effects of intracerebroventricular injections of 5,6-DHT on the development and maintenance of hypertension in spontaneously hypertensive rats has been investigated. 5,6-DHT, injected into 6 week old rats, retarded the development of hypertension for at least 6 weeks; this effect was not accompanied by inhibition of the pressor response produced by stimulation of the total peripheral sympathetic outflow. 5,6-DHT, injected into 14--15 week old rats with established hypertension, produced a short-lived fall in blood pressure. These findings suggest that central 5-HT neurones are involved in the development of hypertension in spontaneously hypertensive rats. Spontaneously hypertensive rats Intracerebroventricular
5,6-Dihydroxytryptamine
1. I n t r o d u c t i o n T h e role o f brain 5 - h y d r o x y t r y p t a m i n e (5HT) as a f u n c t i o n a l t r a n s m i t t e r in the central nervous s y s t e m is s u p p o r t e d b y e x p e r i m e n t a l evidence (Chase and M u r p h y , 1 9 7 3 ) , and whilst 5-HT secreting n e u r o n e s have b e e n identified in v a s o m o t o r regions o f t h e brain (DahlstrSm and F u x e , 1965; F u x e et al., 1 9 6 8 ) t h e i r role in the regulation o f b l o o d pressure has n o t b e e n established. I n t r a c e r e b r o v e n t r i c u l a r (i.c.v.) administrat i o n o f 5 , 6 - d i h y d r o x y t r y p t a m i n e (5,6-DHT) causes a selective d e s t r u c t i o n o f central 5-HT c o n t a i n i n g nerve terminals ( B a u m g a r t e n et al., 1 9 7 2 c ) with a r e s u l t a n t d e p l e t i o n o f brain 5-HT levels ( B a u m g a r t e n et al., 1971, 1 9 7 2 a ) and r e d u c t i o n o f n e u r o n a l 5-HT u p t a k e (BjSrklund et al., 1 9 7 3 ) . Intracisternally administered 5,6-DHT prevents the d e v e l o p m e n t o f n e u r o g e n i c , b u t n o t renal, h y p e r t e n s i o n in rabbits (Wing and Chalmers, 1 9 7 4 ) , b u t does n o t m o d i f y t h e d e v e l o p m e n t o f DOCA/saline
Blood pressure
h y p e r t e n s i o n in rats (Myers et al., 1974}. In t h e present study, 5,6-DHT administered i.c.v. has b e e n used t o evaluate t h e role o f central 5-HT secreting n e u r o n e s in the d e v e l o p m e n t and m a i n t e n a n c e o f raised b l o o d pressure in s p o n t a n e o u s l y h y p e r t e n s i v e rats.
2. Materials and methods 2.1. A n i m a l s
Male s p o n t a n e o u s l y h y p e r t e n s i v e rats (SHrats) (Japanese strain; O k a m o t o and Aoki, 1963) were used f o r these studies. 2.2. D i r e c t recording o f conscious SH-rats
blood pressure in
U n d e r e t h e r anaesthesia, a p o l y t h e n e cannula was i m p l a n t e d in the a b d o m i n a l a o r t a o f rats, using a m o d i f i c a t i o n o f the m e t h o d described by Weeks and J o n e s (1960). T h e rats
432
were allowed 1--2 days to recover before use. Blood pressure was recorded in the conscious unrestrained animal using a Statham P23 Db pressure transducer; heart rate was counted from the record after increasing the chart speed.
2.3. Stimulation of sympathetic outflow in pithed SH-rats SH-rats were set up in pairs (one control and one treated) for stimulation of the entire sympathetic outflow by the method described by Gillespie and Muir (1967). Each rat received i.v. atropine, 1 mg/kg, and d-tubocurarine, 2 mg/kg, after pithing. Preparations were electrically stimulated for periods of 20 sec every 8 min, by monophasic, square-wave pulses of 0.5 msec duration, at supramaximal voltage, over the frequency range 0.25--8 Hz. Changes in diastolic blood pressure were recorded from the indwelling aortic cannula using a Statham P23 Db pressure transducer.
2.4. Drug treatment with i.c.v. 5,6-DHT 2.4.1. SH-rats, 6 weeks old (95--105 g), received an i.c.v, injection of 5,6-DHT, 25 pg, or vehicle, 10 pl of 0.9% w/v saline containing 0.1 mg/ml ascorbic acid, in a manner similar to that described for mice by Haley and McCormick (1957). A 0.5 ml Hamilton syringe with a No. 25 needle was used, together with a No. 20 needle as guide tube. Injections were made to a depth of 3.5 mm in animals lightly anaesthetized with ether. The accuracy of the method was verified in other young rats by location of injected dye in the lateral ventricle. After injection animals were housed in groups of 8 until they attained the required age. At age 8, 10 and 12 weeks, the blood pressure and heart rate (see 2.2.) and body weight of groups of treated and control rats were measured and, on the same day, the animals were pithed and stimulation of the sympathetic outflow was performed (see 2.3.).
R.E. BUCKINGHAM ET AL.
Additionally the blood pressure and heart rate of 6 week old rats were measured by the direct recording method (see 2.2. ). 2.4.2. SH-rats, 14--15 weeks old (260-310 g) received i.c.v, injections via a cannula implanted 3 or 4 days previously according to the method of Hayden et al. (1966). Blood pressure and heart rate were recorded (see 2.2.) in the same group of animals at 24 hr intervals on 5 days {days 0--4). 5,6-DHT, 50 pg i.c.v., or vehicle, 20 pl of 0.9% w/v saline containing 0.1 mg/ml ascorbic acid, was injected immediately after zero time recordings on day 0. In a further group of animals blood pressure and heart rate were recorded at hourly intervals for 4 hr after injection.
2. 5. Drugs used 5,6-Dihydroxytryptamine creatinine sulphate (kindly supplied by Dr. Langemann, F. Hoffman--La Roche, Basle) was dissolved in 0.9% w/v saline containing 0.1 mg/ml ascorbic acid. Doses are expressed as the base. Results are expressed as means + standard error of the mean. Data were analysed using Student's ttest and significance of difference taken at p ~< 0.05.
3. Results
3.1. Effect of pretreatment with 5,6-DHT (25 pg i.c.v.) on the development o f hypertension 2, 4 and 6 weeks after treatment of 6 week old rats with 5,6-DHT, the blood pressure of treated rats was significantly lower than that of age-matched control rats treated with vehicle. At each age the blood pressure of control animals was significantly higher (14--20 mm Hg) (p <: 0.01 at 8 weeks; p < 0.001 at 10 and 12 weeks) than that of treated rats (fig. 1A). Whilst the blood pressure of control rats significantly increased by 18 mm Hg from age 6--8 weeks and by 42 mm Hg from age 6--12 weeks, the blood pressure of 8 week old treated rats was not significantly different from
5,6-DIHYDROXYTRYPTAMINE AND BLOOD PRESSURE
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433
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8
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Age(Weeks)
Fig. 1. Change in (A) mean arterial pressure (BP mm Hg,), (B) heart rate (HR beats/min), (C) body weight (Wt g), with age (weeks), of spontaneously hypertensive rats treated at 6 weeks age with 5,6-DHT~ 25 pg i.c.v. (~ --) or with 10 ~l vehicle (© ©). Groups of 9--12 animals were used; vertical bars extend to S.E.M.
t h a t o f 6 w e e k old rats, a n d at 12 w e e k s age t h e b l o o d pressure of t h e t r e a t e d g r o u p was 22 m m Hg higher t h a n t h a t o f 6 w e e k old rats. T h e h e a r t r a t e o f 8, 10 and 12 w e e k old c o n t r o l and t r e a t e d rats was significantly lower (p < 0.01 at each age) t h a n t h a t o f 6 w e e k old rats; at each age the h e a r t r a t e o f t r e a t e d rats was n o t significantly d i f f e r e n t f r o m t h a t o f a g e - m a t c h e d c o n t r o l a n i m a l s (fig. 1B). A t age 8, 10 a n d 12 weeks t h e b o d y w e i g h t o f t r e a t e d rats was significantly less ( 1 6 - - 3 5 g) (p < 0.01 at each age) t h a n t h a t o f a g e - m a t c h e d c o n t r o l animals. T h e b o d y weights o f 12 w e e k old c o n t r o l and t r e a t e d rats were r e s p e c t i v e l y 156 a n d 140 g greater t h a n t h o s e of 6 w e e k old rats (fig. 1C). T h e d a t a in figs. 1 A and 1C s h o w t h a t f o r t h e s a m e level o f b l o o d pressure in c o n t r o l and t r e a t e d rats, t h e t r e a t e d rats are n o t o n l y o l d e r t h a n c o n t r o l rats b u t also have higher b o d y weight. F o r e x a m p l e , for a b l o o d pressure level o f 120 m m Hg in b o t h groups, t h e b o d y w e i g h t o f c o n t r o l rats ( 1 0 8 g) is 30 g less t h a n t h a t o f t r e a t e d rats; similarly at 142 m m Hg t h e b o d y weight o f c o n t r o l rats ( 2 0 4 g) is 46 g less t h a n t h a t o f t r e a t e d rats.
3.2. Effect o f pretreatment with 5,6-DHT (25 lag i.c.v.) on neuronally induced pressor responses in pithed rats In a g e - m a t c h e d t r e a t e d a n d c o n t r o l rats, t h e f r e q u e n c y - d e p e n d e n t increases in diastolic
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Fig: 2. Relationship between increase in diastolic blood pressure (A BP mm Hg) and frequency of stimulation (Hz) of the spinal cord of pithed rats (age 8 weeks, [circles] and age 12 weeks [triangles] ) in animals pretreated at 6 weeks age with 5,6-DHT, 25 /2g i.c.v. ( e - - - - - 4 ) and (A. . . . . ~), or with 10 #l vehicle i.c.v. (o ©) and (~/x). Groups of 10 animals were used; vertical bars extend to S.E.M.
434
R.E. B U C K I N G H A M E T AL.
blood pressure produced by stimulation of the entire sympathetic outflow in pithed rats were not significantly different. Fig. 2 shows the relationship with frequency of the pressor responses evoked by neuronal stimulation in treated and control rats aged 8 and 12 weeks; similar results were obtained using 10 week old rats. Fig. 2 also shows that, other than stimulation at 0.25 and 0.5 Hz, the pressor responses to neuronal stimulation in 12 week old control rats were significantly higher (p < 0.001) than those evoked in 8 week'old rats; a similar significant difference existed for pressor responses in 10 and 12 week old rats.
10
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3.3. Effect of pretreatment with 5,6-DHT (50 pg i.c.v.) on the blood pressure of SH-rats with established hypertension 5,6-DHT significantly reduced (p < 0.001) the blood pressure of 14--15 week old SH-rats for at least 4 hr after injection; heart rate was significantly increased (p < 0.01) in treated animals 2--4 hr after injection (fig. 3). In a further experiment when measurements were made at 24 hourly intervals for 4 days after injection (fig. 4) the blood pressure and heart rate of treated animals did not differ from those of control rats.
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Fig. 3. T i m e course ( h r ) o f c h a n g e in (A) m e a n arterial b l o o d pressure (A BP m m Hg) a n d (B) h e a r t rate (A HR b e a t s / m i n ) o f 1 4 - - 1 5 w e e k old s p o n t a n e o u s l y h y p e r t e n s i v e rats following 5,6-DHT, 50 pg i.c.v. (~ e ) or 20 #l vehicle i.c.v. (o o). T h e initial m e a n arterial pressure a n d h e a r t r a t e o f each g r o u p were, respectively, for t r e a t e d r a t s 171 + 3 m m Hg a n d 381 + 8 b e a t s / m i n and, for u n t r e a t e d rats 170 +- 3 m m Hg a n d 388 -+ 10 b e a t s / m i n . G r o u p s o f 15 a n i m a l s were used; vertical bars e x t e n d t o S.E.M.
Fig. 4. T i m e course (days) o f (A) m e a n arterial pressure (BP m m Hg) a n d (B) h e a r t r a t e ( H R b e a t s / m i n ) of 1 4 - - 1 5 week old s p o n t a n e o u s l y h y p e r t e n s i v e rats t r e a t e d o n d a y 0 with 5,6-DHT, 50 pg i.c.v. (-, ) (n = 9) or 20 pl vehicle i.c.v. (o o) (n = 11). G r o u p s o f 9 - - 1 1 animals were used; vertical bars e x t e n d t o S.E.M.
5,6-DIHYDROXYTRYPTAMINE AND BLOOD PRESSURE 4. Discussion In this investigation, 5,6-DHT administered i.c.v., has been shown to retard, but not prevent, the development of hypertension in SHrats (Japanese strain) for at least 6 weeks, and to produce a short-lived fall in blood pressure in SH-rats with established hypertension. The doses used in this study have been shown by Baumgarten et al. (1971) to produce a marked and long-lasting reduction in brain 5-HT levels in the rat. Baumgarten et al. (1972c) showed that these effects of 5,6-DHT were the result of selective destruction of indoleamine containing nerve terminals. The retardation of the development of hypertension in our experiments suggests that 5-HT secreting neurones are involved in the central regulation of blood pressure in SH-rats; our findings would be consolidated by data showing the effect of 5,6-DHT in depleting 5-HT levels in the central nervous system of SH-rats. However, as Chalmers (1975) points out, the value of such studies is limited. Earlier studies showed that 6-hydroxydopamine (6-OHDA) administered i.c.v, to rats, produces a widepread destruction of the nerve terminals of central noradrenergic neurones (Uretsky and Iversen, 1970). In other studies 6-OHDA administered i.c.v, has been shown to inhibit the development of DOCA/saline, renal and spontaneous hypertension in the rat (Finch et al., 1972; Haeusler et al., 1972). In the rabbit 6-OHDA administered intracisternally, has been shown to inhibit the development of neurogenic and renal hypertension (Chalmers and Reid, 1972; Lewis et al., 1973). In recent studies Wing and Chaimers (1974) observed that intracisternal administration of 5,6-DHT produced a fall in blood pressure in normal and neurogenically hypertensive rabbits, and prevented the development of neurogenic, but not renal, hypertension in this species. In contrast, the development and maintenance of DOCA/saline hypertension in the rat were unaffected by intracisternally administered 5,6-DHT (Myers et al., 1974). These
435
observations, together with the results of our own studies, tend to suggest that unlike 6OHDA, 5,6-DHT demonstrates some degree of selectivity in its effect on the development of neurogenic hypertension in the rabbit and spontaneous hypertension in the rat. Wing and Chalmers (1974) suggested that the selective destruction of 5-HT containing bulbospinal nerves by 5,6-DHT in the rabbit interferes with the baroreceptor reflex arc and hence the development of neurogenic hypertension. It is possible that the retardation of the development of spontaneous hypertension in rats by 5,6-DHT which we have observed may be the result of an analogous mechanism. The failure of 5,6-DHT to reverse the raised blood pressure in SH-rats with established hypertension, suggests that 5-HT secreting netlrones may be involved in initiating the hypertension in young SH-rats, b u t may not play a significant role in maintaining the elevated pressure. A similar role has been ascribed to central noradrenergic neurones by Haeusler et al. (1972). This being so, there exists the possibility of a dual mechanism of initiation of hypertension in SH-rats. Such a dual control of peripheral sympathetic nerves and arterial blood pressure by bulbospinal nerves in the rabbit has been suggested by Chalmers (1975). It seems unlikely that an effect on brain catecholamines could contribute to the observed effects of 5,6-DHT in retarding the development of hypertension in SH-rats. Baumgarten et al. (1971) found that a rapid reduction in whole brain catecholamine levels followed the i.c.v, administration of 75 pg 5,6-DHT to normotensive rats. Normal catecholamine levels were, however, restored within 2--4 days of the injection. Furthermore the failure of 5,6-DHT to modify the increase in neuronally mediated pressor responses that occurred with age in SH-rats suggests that neither inhibition of peripheral sympathetic functional capacity nor the reduction in b o d y weight gain contributed to the impaired development of hypertension in these rats.
436
The acute fall in blood pressure elicited by i.c.v. 5,6-DHT in 14--15 week old SH-rats may be due to the initial release of endogenous brain noradrenaline and/or 5-HT (Baumgarten et al., 1971) or to the direct stimulant effect of 5,6-DHT on 5-HT receptors (Baumgarten et al., 1972b). There is a growing body of evidence to support a relationship between central 5-HT receptor stimulation and a fall in blood pressure. Bhargava and Tangri {1959) observed that i.c.v. 5-HT produces a marked fall in blood pressure in anaesthetised cats. Baum and Shropshire (1975) have recently demonstrated that i.v. 5-hydroxytryptophan (5-HTP), a precursor of 5-HT, given after peripheral decarboxylase inhibition, produces a progressive reduction in blood pressure and heart rate in anaesthetised cats. This effect is associated with a dose-dependent reduction in spontaneous electrical activity of cardiac, renal and splanchnic nerves. In contrast Henning and Rubenson (1971) showed that, in conscious rats, the hypotensive response to systematically administered 5-HTP is largely prevented by inhibition of peripheral decarboxylase. The acute antihypertensive response to i.c.v. 5,6-DHT in our experiments is unlikely to be peripherally mediated since i.v. 5,6DHT produces a pressor response in pithed rats (Baumgarten et al., 1972b).
Acknowledgement The authors are grateful to Mr. D.T. Burden for his technical assistance.
References Baum, T. and A.T. Shropshire, 1975, Inhibition of efferent sympathetic nerve activity by 5-hydroxytryptophan and centrally administered 5-hydroxytryptamine, Neuropharmacol. 14, 227. Baumgarten, H.G., A. BjSrklund, L. Lachenmayer, A. Nobin and U. Stenevi, 1971, Long-lasting selective depletion of brain serotonin by 5,6-dihydroxytryptamine, Acta Physiol. Scand., Suppl. 373. Baumgarten, H.G., K.D. Evetts, R.B. Holman, L.L.
R.E. BUCKINGHAM ET AL. Iversen, M. Vogt and G. Wilson, 1972a, Effects of 5,6-dihydroxytryptamine on monoaminergic neurones in the central nervous system of the rat, J. Neurochem., 19, 1587. Baumgarten, H.G., M. GSthert, H.G. Schlossberger and P. Tuchinda, 1972b, Mechanism of pressor effect of 5,6-dihydroxytryptamine in pithed rats, Naunyn-Schmiedeb. Arch. Pharmacol. 274, 375. Baumgarten, H.G., L. Lachenmayer and H.G. Schlossberger, 1972c, Evidence for a degeneration of indoleamine containing nerve terminals in rat brain induced by 5,6-dihydroxytryptamine, Z. Zellforsch. 125, 553. Bhargava, K.P. and K.K. Tangri, 1959, The central vasomotor effects of 5-hydroxytryptamine, Brit. J. Pharmacol. Chemoterap. 14, 411. BjSrklund, A., A. Nobin and U. Stenevi, 1973, Effects of 5,6-dihydroxytryptamine on nerve terminal serotonin and serotonin uptake in the rat brain, Brain Res. 53, 117. Chalmers, J.P., 1975, Brain amines and models of experimental hypertension, Circulation Res. 36, 469. Chalmers, J.P. and J.L. Reid, 1972, Participation of central noradrenergic neurones in arterial baroceptor reflexes in the rabbit. A study with intra-cisternally administered 6-hydroxydopamine, Circulation Res. 31,789. Chase, T.N. and D.L. Murphy, 1973, Serotonin and central nervous system function, Ann. Rev. Pharmacol. 13,181. DahlstrSm, A. and K. Fuxe, 1965, Evidence for the existence of monoamine-containing neurons in the central nervous system. I. Demonstration of monoamines in the cell bodies of brain stem neurones, Acta Physiol. Scand. 62, Suppl. 232. Finch, L., G. Haeusler and H. Thoenen, 1972, Failure to induce experimental hypertension in rats after intraventricular injection of 6-hydroxydopamine, Brit. J. Pharmacol. 44, 356. Fuxe, K., T. HSkfelt and U. Ungerstedt, 1968, Localisation of indolealkylamines in CNS, Advan. Pharmacol. 6A, 235. Gillespie, J.S. and T.C. Muir, 1967, A method of stimulating the complete sympathetic outflow from the spinal cord to blood vessels in the pithed rat, Brit. J. Pharmacol. Chemotherap. 30, 78. Haeusler, G., L. Finch and H. Thoenen, 1972, Central adrenergic neurones and initiation and development of experimental hypertension, Experientia 28, 1200. Haley, T.J. and W.G. McCormick, 1957, Pharmacological effects produced by intracerebral injection of drugs in the conscious mouse, Brit. J. Pharmacol. Chemotherap. 12, 12. Hayden, J.F., L.R. Johnson and R.P. Maickel, 1966, Construction and implantation of a permanent
5,6-DIHYDROXYTRYPTAMINE AND BLOOD PRESSURE cannula for making injections into the lateral ventricle of rat brain, Life Sci. 5, 1509. Henning, M. and A. Rubenson, 1971, Effects of 5-hydroxytryptophan on arterial blood pressure, body temperature and tissue monoamines in the rat, Acta Pharmacol. Toxicol. 29, 145. Lewis, P.J., J.L. Reid, J.P. Chalmers and C.T. Doilery, 1973, Importance of central catecholaminergic neurons in the development of renal hypertension, Clin. Sci. 45, 115. Myers, M.G., J.L. Reid and P.J. Lewis, 1974, The effect of central serotonin depletion on DOCA/saline hypertension "'l the rat, Cardiovascular Res. 8, 806.
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Okamoto, K. and K. Aoki, 1963, Development of a strain of spontaneously hypertensive rats, Jap. Circulation J. 27,282. Uretsky, N.J. and L.L. Ivecsen, 1970, Effects of 6-hydroxydopamine on catecholamine containing neurones in the rat brain, J. Neurochem. 17,269. Weeks, J.R. and J.A. Jones, 1960, Routine measurement of arterial pressure in unanaesthetised rats, Proc. Soc. Exptl. Biol. Med. 104, 646. Wing, L.M.H. and J.P. Chalmers, 1974, Participation of central serotonergic neurones in the control of the circulation of the unanaesthetised rabbit, Circulation Res. 35, 504.