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Central serotonergic participation on blood pressure regulation
Gen. Pharmac. Vol. 13. pp. 427 to 432. 1982 Printed in Great Britain All rights reserved
0306-3623:82 050427-06503.00:0 Copyright © 1982 Pergamon Press Ltd
CENTRAL SEROTONERGIC PARTICIPATION ON BLOOD PRESSURE REGULATION AGUSTfN Jos~' RAMIREZ,* ELBA CHEMERINSKI and MARIA AMELIA ENERO lnstituto de Investigaciones Farmacologicas. CONICET, Cfitedra de Farmacologia, Facultad de Farmacia y Bioquimica, UBA, Junin 956, 5" Piso, 1113 Buenos Aires. Argentina (Received 22 March 1982)
Abstract--1. The changes on blood pressure induced by pharmacological handling of serotonergic systems were studied in normotensive anesthetized rats. Administration of 5-hydroxytryptophan (5-HTP) 15, 10 and 30 mg/kg i.v.) produced a dose-dependent decrease in mean blood pressure without significative effect on heart rate. 2. Inhibition of either peripheral L-aminoacid DOPA decarboxylase or monoamine oxydase enzymes increased this hypotensive effect. 3. Methysergide, a serotonergic antagonist, prevented the hypotension induced by 5 0 H - T P in animals whose peripheral decarboxylase was inhibited. 4. Fenfluramine, a serotonergic releasing drug, produced a decrease in both blood pressure and heart rate. These effects were prevented by inhibiting fenfluramine uptake or by serotonin (5-HT) receptor blockade. 5. The hypotensive action induced by 5-HTP was not affected when opioid, dopaminergic or histaminergic receptors were blocked. 6. In animals injected intracisternally with a serotonergic neurotoxine a selective destruction of serotonergic terminals of spinal cord was obtained. 7. In these animals the dose-response curve relating hypotensive effect induced by a direct serotonergic agonist showed a significative shift to the left when compared with control group, suggesting the existence of supersensitivity. 8. Our results show that increases in central serotonergic activity produce a hypotensive effect in normotensive anesthetized rats. The receptor involved in this action could be localized in spinal cord.
INTRODUCTION
cio & Robson, 1975) which could be mediated by a decrease in the preganglionic sympathetic activity (Franz et al., 1975; Tadepalli et al., 1977). In contrast, Chalmers (1975) observed that the decrease in serotonergic bulbo-spinal neurons of hypertensive sino-aortic denervated rabbits reduced blood pressure back to control values. Additionally, when central serotonin levels were reduced, by para-chlorophenylalanine (p-CPA) administration, a fall in blood pressure was obtained. These results suggest that central serotonergic fibers are responsible for a pressor response. Fenfluramine, a serotonergic releasing drug, showed an antihypertensive effect in men, possibly through a reduction of the central sympathetic outflow as a consequence of enhanced brain serotonin neural function (Tuomilheto et al., 1977; De La Vega et al., 1977). In the present study we have further analyzed the effects on cardiovascular functions of pharmacological agents known to affect on serotonergic transmission.
The participation of serotonergic pathways in the central nervous system regulating the cardiovascular function has been a subject of numerous studies. In early works Bogdanski et al. (1958) observed that the administration of 5-hydroxytryptophan (5-HTP), a serotonin precursor, in dogs, induced hypertension a n d tachycardia. The authors suggested that these actions could be mediated by several mechanisms localized in the periphery and in the central nervous system (CNS). In addition, it was demonstrated that serotonergic neurons localized in brain-stem project their axons to the sympathetic lateral column of the spinal cord accompanying sympathetic descending fibers originated from adrenergic neurons (DahlstriSm & Fuxe, 1965). The possibility of these neurons regulating the vasomotor activity was previously suggested by different authors [Lim et al., 1938; Alexander, 1946; Carlsson et al., 19641. Subsequent investigations showed not always coincidental results. Some authors reported that 5 - H T P by its conversion to serotonin (5-HT) in the CNS, produced a hypotensive effect (Ito & Schanberg, 1972; Florez & Armijo, 1974; Antonac-
METHODS
* Present address: Centro di Fisiologia Clinica e Ipertensione, Clinica Medica IV. Universitfi di Milano, 20122 Milano, Viii F. Sforza 35, Italy. 427
Wistar female rats weighing 120-180 g were anesthetized with urethane 1.2 g/kg. (i.p.I. The trachea was cannulated and the arterial blood pressure was measured from carotideal cannula with a Statham P 23-AC transducer and recorded with a Grass model 7 polygraph.
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Mean arterial" blood pressure "*as calculated according t,,I tile follov, ing fornlula: I 3 (sistolic pressure-diastohc pressure)- diastolic pressure. Heart rate was i n e a s u r e d with an [:~.K.Ck-Tachograpll preanlpliiier (Grass model 7 P4). A femoral ~ein was also cannulated for s~stemic drug administration. IntracL~ternal 5 7 dihydrox.ttr.ffmmnnc (5 7 Dtt 7"1 admini.sIra/ion Female Wistar rats weighing 150 180g were administered desipramine (DMI). 25 mgkg i.p.. I hr before intracisternal administration, of 5 7 DHT. After recei`.'ing a light ether anesthesia the animals '*ere placed in a stand similar to that described by Jeffers & Griflhh (1967). Puncture site ~,as localized in a depression between the protuberance and the spree of the atlas, directly caudal to the occipital protuberance. Once the needle ',',as introduced. 25 pg of 5- 7 DHT in 2 !d saline sc,lution containing 0.2 mg ml of ascorbic acid was injected. Another group of rats was identically treated but onl} vehicle "*as injected .%'~'ro/otlitt tl~.s~l)'s
After sacriticing the animals by decapitation the brains were quickl,, removed The brain areas: midbrain, pons and medulla obhlngata were dissected out at 4 C. "The spinal cord was also dissected and divided into three sections: cervical, thoracic and lumbar. All samples ,.,,ere immediately frozen at - 6 0 C until the assa`, v,as carried out (no more than 18 hr later). The tissues ,,,,'ere homogenized it] butanol acid mixture and 5 HT was assayed fluorometrically according to the procedure described by Curzon & Green (19701. Stati.stical methods All values herein reported represent mean _+ SEM. The statistical significance of differences between groups were calculated by means of Student's t-Test (Snedecor & Cochran. 1967). Drug~ The following drugs were used: 5 Hydroxytryptophan propionic acid: 5 7 dihydroxylryptamine bimaleale and 5-methoxy .\'-N dimethyltryptamine {Sigma): MK 486 [Merck. Sharp & Dohme): pargyline chloride (Abbot): methysergide maleate (Sandoz): fenfluramine (Newark): fluoxetine hydrochloride [Lilly); burimamide 1S. K. F. Laboratories); naloxone hydrochloride (Endo): pyrilamine maleate (Rhone-Poulene): desmethylimipramine chloride (Geigy) and pimozide (Janssen). RESII.FS
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Fig. 1. Effects on mcan blood pressurc induced by 5-ttTP administration. 5-tITP "*as administered i.v. in doses of: 5 (O): 10 I&)and 30 (i)mg:kg. Shaded area shows the mean blood pressure `.'ariations along time of animals that received saline solution (control groupl. Pre: indicates basal mean blood pressure values before treatment. Filled symbols show significance from control group (P -< 0.05). onist, methysergide (3 mg.kg i.v.) administrated 5 min before the serotonin precursor [Fig. 2). On the other hand, the serotonergic releasing drug, fenfluramine (5 mg:kg i.v.) produced a decrease in the mean blood pressure similar to that obtained with 10 m g k g 5-HTP [Fig. 3). This effect was prevented by injecting 5 rain before either fluoxetinc, a serotonin uptake inhibitor (2mg kg i.v.) or methysergide (3 mg. kg) (Fig. 3). Probable participation o f other neurotransmitter.s in the 5 - H T P induced cardiovascular response.s To study the probable interaction of serotonin with other neural systems, the responses to 5-HTP were evaluated in rats pretreated with different antagonists. Naloxone (5 mg/kg) failed to antagonize the hypotensive action induced by the serotonin precursor when injected 5 rain before it (Fig. 4a). Similarly, the blockade of either histamine Ha or Hz receptors with mOO[ I
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I.ig 2. F!ffect on the hypotensi`.,e action of 5-HTP induced by MK 486: Pargyline and methysergide. {-} indicates control blood pressure values. ( - ) maximal blood pressure effect induced by 5-HTP 15 mg kg) "*ithout pretreatment or previously administered v,ith either MK 486 (30rain before) or pargyline 118 h before). MK 486 + Methv.: animals pre`.'iously administered with MK 486 130 min before 5-HTP) and methysergide (Methy. 25 min after MK 486). *P < 0.05.
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Fig. 3. Hypotensive action of fenfluramine. ( - I indicates control mean blood pressure. ( + t maximal hypotensive action of fenfluramine alone or in animals pretreated with fluoxetine (Fluox.) or methysergide (Methy.). Both drugs were administered 5 min before fenfluramine. *P < 0.05. b u r i m a m i d e (2 mg/kg i.v.I or pyrilamine (2 mg/kg i.v.I respectively did not affect the decrease in blood pressure produced by 5 - H T P (Fig. 4c and d). The d o p a m i n e receptor blocking drug, pimozide (3 mg/kg i.v.) induced a slight decrease in blood pressure basal values that did not interfere with the hypotensive action of 5 - H T P (Fig. 4b).
Effeet.~ on serotonin lerels and .serotonergic h)'potensire action o]5-7 D H T A selective depletion of endogenous serotonin levels was obtained by injecting intracisternally 25/~g of serotonergic neurotoxin 5-7 DHT. In the spinal cord 5-HT levels decreased 40-50% while in the brain-stem only 10°', (Table 1). Thus, this depletion would indicate a partial destruction of serotonergic terminals in the spinal cord. according to Trulson et al. (1976). To study how these reductions on the 5-HT levels of the spinal cord affect the responses to a direct 5-HT agonist, the animals were injected intracisternally 7 days before the experiment with 5-7 DHT.
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Fig. 5. Dose-response curve relating to 5 Me O-DMT effects on mean blood pressure. 5 Me O-DMT was given i.v. to either saline or 5 7 DHT intracisternally injected rats. Ordinates shows differences in mean blood pressure. abscissa : Doses of the serotonergic direct agonist in ,~g/kg. 0 : Control group (Saline solution intracisternal. 7 days beforeJ. O: Denervated animals (5 7 DHT intracisternal, 7 days beforc). *P < 0.05. Figure 5 shows that in the control group a hypotensive effect was obtained with 1 and 3 mg/kg of 5 methoxy N - N dimethyltryptamine (5 Me O - D M T ) whereas in the denervated group a shift to the left of approximately one logarithmic unit in the dose-response curve was observed.
Effects on heart rate The treatments used affected the heart rate in different ways. Doses of 10 and 30 mg/kg of 5 0 H - T P induced a slight bradycardia. 10mg/kg: lower from 413 + 22 b p m to 372 + 20 b p m ; 3 0 m g / k g from 423 + 17 to 391 +__ 13 b p m (in b o t h groups n = 4). None of the receptor blockers used affected the heart rate (Table 2A). In contrast, when fenfluramine was administered, a significant decrease in the heart rate was obtained. This action was prevented by pretreating the animals with fluoxetine or methysergide (Table 2B).
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Fig. 4. Effect induced by different antagonists on the hypotcnsivc action of 5-HTP. ( - ) indicates basal mean blood pressure. (~-1 maximal hypotensive action of 5-HTP (5 mg/kg). Pretreatments: (al Nal.: naloxone 5mg/kg. (b) Pim.: pimozide 3mgkg. (c) Bur.: burimamide 2mg/kg. (dl Pir.: pyrilamine 2 mg:kg. All the antagonists were givcn intravenously 5 min before 5-HTP. *P < 0.05: compared ~,ith previous basal value. A: P < 0.05: compared with control group.
43(1
A c ; t s l i y Jos(; RAMI'RI:Z el O/.
Table 1. Serotonin le~.els in rats inJected either with sahne solution or 5 intracisternally
DHT
5-11T lig.g Iwet. ~tl" ('ontrol
Area Midbrain Pons Medulla oblongata Cervical spinal cord Thoracic spinal cord Lumbar spinal cord
0.87 1.11 0.83 1.63 1.16 2.44
5- 7 1)l-IT
+_ 0.03 _+ 0.05 + 0.02 _+ 0.06 _+ 0.04 _+ 0.27
0.79 0.99 0.87 0.85 0.68 1.15
_+ 0.03 5 0.02 : 0.10 -- 0.04* ..- 0.05* +_ 0.19"
Reduct? -x - 11 ~-6 -47 - 41 -53
Per cent of the decrease in serotonin levels induced by administering 5 7 DHT I.C.M. 7 days previous. *P < 0.05; n = 4. DISCUSSION T h e s e r o t o n i n p r e c u r s o r 5 - H T P p r o d u c e d a dosed e p e n d e n t decrease in the m e a n b l o o d pressure w h e n a d m i n i s t e r e d intravenously to n o r m o t e n s i v e anesthetized rats, being the m i n i m u m effective dose of 5 mg/kg. After a d m i n i s t r a t i o n of M K 486, an inhibitor o f peripheral decarboxylase, the s h o r t lasting fall in b l o o d pressure p r o d u c e d by 5 m g / k g o f 5 - H T P was not only e n h a n c e d but also r e m a i n e d significantly lower than c o n t r o l values for m o r e than 30 min. This effect suggests an increased availability o f 5 - H T P to the CNS. This interaction, a n d the p o t e n t i a t i o n o b served in a n i m a l s p r e t r e a t e d with a M A O I , s u p p o r t s the idea that, to p r o d u c e h y p o t e n s i o n , 5 - H T P should be previously c o n v e r t e d to s e r o t o n i n in the CNS. F u r t h e r m o r e , an a d d i t i o n a l evidence a c c o u n t i n g for the s t i m u l a t i o n o f a s e r o t o n e r g i c r e c e p t o r is p r o v i d e d
by the fact that m e t h y s e r g i d e p r e v e n t e d the reduction in b l o o d pressure i n d u c e d by 5 - H T P . T h e s e results agree with those r e p o r t e d by o t h e r a u t h o r s in different species like cats IFl6rez Armijo. 1974: F r a n z et al.. 1975: Tadepalli et al., 19771 and dogs ( A n t o n a c c i o & R o b s o n , 1973, 1975). lto & S c h a n b e r g (1972) r e p o r t e d that the inhibition o f the central t r y p t o p h a n hydroxylase in rats prod u c e d an increase in b l o o d pressure c o r r e l a t e d with the s e r o t o n e r g i c depletion. After the injection of 5 - H T P , that b y p a s s e s the enzyme, the b l o o d pressure r e t u r n e d to basal values. M o r e o v e r . p r o v o k i n g brainstem lesions surgically the 5 - H T P site o f action was localized in the rostral p o r t i o n o f the medulla o b l o n gala. O n the o t h e r hand, H e n n i n g & R u b e n s o n (1971j r e p o r t e d that 5 - H T P a d m i n i s t e r e d to c o n s c i o u s rats p r o d u c e d a significant fall in b l o o d pressure that was peripheral in origin since w h e n the peripheral decar-
Table 2(A). Effects on heart rate induced by different drug administration
Pretreatmenl + M K 486 + Parg~line + Naloxone Pinlozide + Burimarnide + P,,rilanaine.
Ileart rate ibeats.:mm) 5-HTP maximal Basal value ~ effect b 359 354 382 332 364 368 36(1 384 364 368 .~47
+ 22 4_ 24 _+ 32 !. 34 _+ 17 i 26 ÷8 + 28 ! 33 _+ 19 ± 25
343 319 415 302 355 353 346 343 358 372 345
± 26 4=_8 _+ 40 ~ 23 t- 5 _+ 5 j 9 ~ 36 .l 33 -.- 20 _t _:,~-
n 3 3 3 5 5 4 4 5 5 3 3
Table 2(B).
Pretreatment -Fluoxetine -Methysergide
Heart rate (beats.min) Fenfluramine maximal Basal value s effect h 392 _+ 14 390 + 8 365 + 15
329 ! 18" 368 + 29 345 _+ 34
n 7 5 4
Shown are the maximal effects induced by (A) 5-HTP (5 mg:kgk or (B} fenfluramine (5 mg:kg) given to animals that did not receive, or received different pretreatrnents. J Pretreatments effect on basal heart rate; and t, on either the 5-1tTP or fenfluramine maximal effect. *P < 0.05.
Serotonin on blood pressure boxylase was inhibited by pretreating the animals with MK 486, neither hypotensive effect nor 5-HT increased levels in CNS were observed. In a similar way, when the animals were pretreated with nialamide, a MAOI. and MK 486 once again no significant action of 5-HTP was obtained. However, according to Fuxe et al. (1971) the ability of the 5-HT neurons to take up and accumulate 5-HT in the presence of 5-HTP is low in spite of the large amounts of 5-HTP present in the brain after extracerebral decarboxylase inhibition: the specificity for the 5-HT neurons is obtained with 20-100mg/kg of 5-HTP. Thus, the discrepancies in Henning & Rubenson results could be due to the fact that the authors mentioned above used higher 5-HTP doses than us. Moreover, Horita & Hamilton [1970) studied the central hyperthermic action induced by 5-HTP in rabbits; doses from 35 50 mg/kg of 5-HTP produced a dose-dependent hypcrthermia. Brain 5-HT levels raised 57°~, when 35 mg/kg (i.v.t of 5-HTP was injected. In animals pretreated with the peripheral decarboxylase inhibitor, 35 mg/kg of 5-HTP, produced marked increase in temperature and a 194"~o augment in the brain 5-HT concentration. These data further support the idea that the higher doses of 5-HTP (200 mg/kg i.v.) used by Henning & Rubenson (1971 ) masked their results. The possibility that the hypotensive action could be mediated by displacement of neurotransmitters other than serotonin is open, since 5-HTP could be decarboxylated in sites other than serotonergic neurons like catecholaminergic neves or vascular endothelium (Fuxe et al., 1971; Pletscher & Bartholini, 1971; Yunger & Harvey, 19761. However, this possibility is discarded since the blockadc of serotonergic receptors by methysergide antagonized the effect of 5-HTP. Moreover, fenfluramine, a serotonergic releasing drug, also produced hypotension and this effect was antagonized by either fluoxetine or methysergide. All these results indicate that the decrease in the mean blood pressure observed after injecting 5-HTP in anesthetized rats is due to its conversion to serotonin in the CNS. Our results blocking morphine, dopamine or histamine receptors show that, the hypotension induced by 5-HTP is due to the stimulation of a serotonergic receptor and does not depend on the existence of a dopaminergic, histaminergic or opioid consecutive pathway. Previous published results (Ito & Schanberg, 1972: Tadepalli et ul., 19771 suggested that the hypotension obtained after serotonergic stimulation is mediated by a bulbo-spinal serotonergic pathway whose origin is localized in the medulla oblongata. This serotonergic action would be mediated by inhibition of preganglionic sympathetic activity. Additionally, the stimulation of mcdullar areas and ~rotonergic descending tibers in cats induced a decrease in mean blood pressure and sympathetic preganglionic activity (Franz et al., 1975). On the other hand, several reports suggested that the hypothalamic serotonergie stimulation in rats observed when administering 5-HT i.c.v. (Nahmod et al., 1978) or the stimulation of the serotonergic mesencephalic nucleus (Smits et al., 1978: Kuhn et al., 1980)
431
induced a hypertensive effect. However, in spite of increasing the brain 5-HT levels a 165~o (with 5-HTP i.c.v, plus a peripheral decarboxylase inhibitor) no changes in the blood pressure were observed (Lin, 1979). From these reports it could be suggested that the serotonergic system may be responsible, depending on the stimulated pathway, for a hypertensive or hypotensive response. Furthermore, Lambert et al. 0978) reported that the effect of 5-HT on blood pressure in rats appeared to be dependent on the manner in which the transmitter is introduced into the brain. Therefore, it is interesting to evaluate which is the effect of the serotonergic stimulation when spinal receptors are supersensitive. In animals pretreated with 5 7 DHT, 7 days before the experiment, a potentiation of the hypotensive effect of 5 Me O DMT was observed. This potentiation indicates the existence of denervation supersensitivity following destruction of bulbo-spinal serotonergic terminals and supportes the idea that these receptors, responsible for the hypotensire action, are localized in the spinal cord. These results could be an evidence supporting Franz et al. 11975) who suggested that 5-HT and noradrenaline descending pathways modulate the preganglionic sympathetic activity. Kuhn et al. (1980) reported a selective pressor effect on the ascending 5-HT neuronal system in rats, by electrical stimulation of the dorsal and median raphe nucleus, which was abolished by rostral transections of the raphe nuclei presumably by interrupting the ascending tract of the 5-HT neuronal system. On the other hand, lto & Schanberg [1972) described a bulbo-protuberancial area which could be responsible for the hypotensive effect observed after 5-HTP administration in tryptophan hydroxylase inhibited rats. Both reports further suggest that in rats 5-HT may participate in pressor and depressor areas involved in the cardiovascular response. In our case, a direct 5-HT agonist (5 Me ODMT) administered to anesthetized rats, induced a lowering of the blood pressure. Therefore, only the hypotensive descending 5-HT neuronal system as being stimulated or expressed. Our results failed to demonstrate any effect of 5-HTP on heart rate. Other authors, however, have shown that this serotonergic precursor produces bradycardia (Antonaccio & Robson, 1973, 1975; FI6rez & Armijo, 1974; Tadepalli et al., 1977). The reasons for these discrepancies are not known. Nevertheless, our results are not in complete contradiction with those of the authors cited above since fenfluramine reduced the heart rate. This effect was mediated by serotonergic stimulation since it was prevented by a 5-HT antagonist or a serotonin uptake blocker. In conclusion, our studies have shown that the increase in the serotonergic activity by injection of: a precursor or an agonist of 5-HT or a serotonergic releasing drug, reduced the blood pressure in the anesthetized rat. This effect could be due to an activation of serotonergic receptors localized in the spinal cord.
A c k n o w l e d . q e m e n t s - This work was supported by a grant from the Consejo Naeional de investigaciones Cientificas y T6cnicas [CONICET).
432
AG~.:ST/N J(>sI~ RAMiRI!Z eta/.
f h e authors wish to thank l)r Francisco J. li. Stefano for reading the manuscript and h~r his helpful criticism of the work. We also greatfully acknowledge Miss Susy Ingala for her technical assistance.
REFERENCES
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and tissue monoanlines in the rat. +Iota pharmac, tuv. 29, 145-154. H(>RII.a, A. & H..\Slll. lOX A. E. I1970) Potentiation of the central actions of 5-hydroxytr~ptophan m rabbits by' In:c-hydrazino-:~-methydopa. ,I. Pharm. I'Ilarn~ac. 22, 389 390. lit> A. & S( tlANBIiRG S. M. 119721 Central ner,,ous :,,?stem mcchanisms responsible for blood pressurc elevation induced by p-chlorophcnylalanine, a. Pharmac. e.\'l,. Ther. 181, 65 74. JEH-I:RS ~d~,'. A. & GRIFI-IIII JR J. Q. 11967) The central nervous system: Cisternal ptlnclure. In lht, Rtit in Laboratory Intesti~lation lEditcd by F,xrrls I-:. J. & Grll.\:Jill J . Q . I p p . 196 199. KtlIN I). M., ~,'ol F W. A. & LtIVI!NBER(.i W. [19801 Pressor effects of electrical stinlulation of the dorsal and median raphe nuclei in anesthetized rats. J. Pharmac. c.'¢p. "lher. 214, 403. 409. LAMBI!RI G . A.. ["RIIil)MAN 1-~.. I'}I'('IIWI:IIZ ti. (~ GliRSIIliN
S. 119781 Involvement of 5-hydrox',tryptamme in the central control of respiration, blood press\ire and heart r a t e ill tile anesthetized rat. .X'euroltharnlacolo~ly 17, 807 813. I.IN M. T. (19791 ISft'ects of brain serotonin aherations on prostagkuldin E t-induced bradycardia in rats..I. Ph,rma~. c.xp. Ther. 208. 232 235. 1.1',1 R. K. S.. W..xy~.; S. C. & YI ('. L. 119381 On the question of myelencephalic sympathetic centers. Clin. J. Phvsi,,I. 13, 61 78. NAtIMOI) V. L.+ I"INKIH MAN N.. BENARRI>('H E. [~. & PlROI A
C. J. 11978! Angiotension regulates release and s)nthesis of serotonin in brain. Science 202, 1091 1093. PI.I rS{'IIIR A. & BARTItOI.INI G. 119711 Selccme rise in brain dopaminc by inhibition of extraccrebral levodopa dccarboxylation. CIm. Phurmac. Thor. 12, 344 352. SMtlsJ. F . M . . \ A X Essl:x H . & S r r t ' , c . ~ r B o t l m r H. A. .1. ( 10781 Serotonin-mediated cardim ascular responses to electrical stimulation of the raphe nuclei m the rat. l.ilc Sci. 23, 173 178. G . W. & ('(>('IlRAN ~V. G . 119671 .Ytoti~tical .Xh'th,Ms The Iov, a State ltni\crsity Press. Anles. Iowa.
SNIIJI('(tR
"l',M)l:l'al I I A. S.. MII.I.S E. & S('I.I,~NBFR(I S. M. 119771 C e n tral depression of carotid barorcccptor prcssor response.
arterial pressure and heart rate b', 5-h)droxy try ptophan: Inlluence of supracollicular areas of the brain. J. I'harmac. exp. Thor. 202, 31l) 319. TRtlSOX M. t[.. tSt:m\\~s F. E. & JA('itBS B. 1. 119761 Bella~ioral e~idence for supcrsensiti~it5 fc,llo'aing destruction of central scrotoncrgic ncr,.e terminaIs b) 5 7 dih~drox.vtryptamine. J. I'harmm. exp. lher. 198, 23 32. +I'uoMII H|.I(> J.. ,~11 IANIIN H. & Jl SI'I:RSlCN .~, 119771 A M u d ' , of the effects of fenlluramine in obese, hypertensi,,c patients treated v.'ith [¢ atdrencrgic blocking age\its. ('u,'r. thor. Res. 22, 821 827. Yt ",a;~u L. M. & II.\~\l', .1. A. 11976} Behavioral effects of t.-5-hydrox+,,tr 3ptophan after destruction of ascending serotonergic palhv.a,,s in the rat: The role of catecholaminergic neurons..I. Pharnuw. e\p. l'her. 196. 3('17 315.
0306-3623:82 050427-06503.00:0 Copyright © 1982 Pergamon Press Ltd
CENTRAL SEROTONERGIC PARTICIPATION ON BLOOD PRESSURE REGULATION AGUSTfN Jos~' RAMIREZ,* ELBA CHEMERINSKI and MARIA AMELIA ENERO lnstituto de Investigaciones Farmacologicas. CONICET, Cfitedra de Farmacologia, Facultad de Farmacia y Bioquimica, UBA, Junin 956, 5" Piso, 1113 Buenos Aires. Argentina (Received 22 March 1982)
Abstract--1. The changes on blood pressure induced by pharmacological handling of serotonergic systems were studied in normotensive anesthetized rats. Administration of 5-hydroxytryptophan (5-HTP) 15, 10 and 30 mg/kg i.v.) produced a dose-dependent decrease in mean blood pressure without significative effect on heart rate. 2. Inhibition of either peripheral L-aminoacid DOPA decarboxylase or monoamine oxydase enzymes increased this hypotensive effect. 3. Methysergide, a serotonergic antagonist, prevented the hypotension induced by 5 0 H - T P in animals whose peripheral decarboxylase was inhibited. 4. Fenfluramine, a serotonergic releasing drug, produced a decrease in both blood pressure and heart rate. These effects were prevented by inhibiting fenfluramine uptake or by serotonin (5-HT) receptor blockade. 5. The hypotensive action induced by 5-HTP was not affected when opioid, dopaminergic or histaminergic receptors were blocked. 6. In animals injected intracisternally with a serotonergic neurotoxine a selective destruction of serotonergic terminals of spinal cord was obtained. 7. In these animals the dose-response curve relating hypotensive effect induced by a direct serotonergic agonist showed a significative shift to the left when compared with control group, suggesting the existence of supersensitivity. 8. Our results show that increases in central serotonergic activity produce a hypotensive effect in normotensive anesthetized rats. The receptor involved in this action could be localized in spinal cord.
INTRODUCTION
cio & Robson, 1975) which could be mediated by a decrease in the preganglionic sympathetic activity (Franz et al., 1975; Tadepalli et al., 1977). In contrast, Chalmers (1975) observed that the decrease in serotonergic bulbo-spinal neurons of hypertensive sino-aortic denervated rabbits reduced blood pressure back to control values. Additionally, when central serotonin levels were reduced, by para-chlorophenylalanine (p-CPA) administration, a fall in blood pressure was obtained. These results suggest that central serotonergic fibers are responsible for a pressor response. Fenfluramine, a serotonergic releasing drug, showed an antihypertensive effect in men, possibly through a reduction of the central sympathetic outflow as a consequence of enhanced brain serotonin neural function (Tuomilheto et al., 1977; De La Vega et al., 1977). In the present study we have further analyzed the effects on cardiovascular functions of pharmacological agents known to affect on serotonergic transmission.
The participation of serotonergic pathways in the central nervous system regulating the cardiovascular function has been a subject of numerous studies. In early works Bogdanski et al. (1958) observed that the administration of 5-hydroxytryptophan (5-HTP), a serotonin precursor, in dogs, induced hypertension a n d tachycardia. The authors suggested that these actions could be mediated by several mechanisms localized in the periphery and in the central nervous system (CNS). In addition, it was demonstrated that serotonergic neurons localized in brain-stem project their axons to the sympathetic lateral column of the spinal cord accompanying sympathetic descending fibers originated from adrenergic neurons (DahlstriSm & Fuxe, 1965). The possibility of these neurons regulating the vasomotor activity was previously suggested by different authors [Lim et al., 1938; Alexander, 1946; Carlsson et al., 19641. Subsequent investigations showed not always coincidental results. Some authors reported that 5 - H T P by its conversion to serotonin (5-HT) in the CNS, produced a hypotensive effect (Ito & Schanberg, 1972; Florez & Armijo, 1974; Antonac-
METHODS
* Present address: Centro di Fisiologia Clinica e Ipertensione, Clinica Medica IV. Universitfi di Milano, 20122 Milano, Viii F. Sforza 35, Italy. 427
Wistar female rats weighing 120-180 g were anesthetized with urethane 1.2 g/kg. (i.p.I. The trachea was cannulated and the arterial blood pressure was measured from carotideal cannula with a Statham P 23-AC transducer and recorded with a Grass model 7 polygraph.
42~
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Mean arterial" blood pressure "*as calculated according t,,I tile follov, ing fornlula: I 3 (sistolic pressure-diastohc pressure)- diastolic pressure. Heart rate was i n e a s u r e d with an [:~.K.Ck-Tachograpll preanlpliiier (Grass model 7 P4). A femoral ~ein was also cannulated for s~stemic drug administration. IntracL~ternal 5 7 dihydrox.ttr.ffmmnnc (5 7 Dtt 7"1 admini.sIra/ion Female Wistar rats weighing 150 180g were administered desipramine (DMI). 25 mgkg i.p.. I hr before intracisternal administration, of 5 7 DHT. After recei`.'ing a light ether anesthesia the animals '*ere placed in a stand similar to that described by Jeffers & Griflhh (1967). Puncture site ~,as localized in a depression between the protuberance and the spree of the atlas, directly caudal to the occipital protuberance. Once the needle ',',as introduced. 25 pg of 5- 7 DHT in 2 !d saline sc,lution containing 0.2 mg ml of ascorbic acid was injected. Another group of rats was identically treated but onl} vehicle "*as injected .%'~'ro/otlitt tl~.s~l)'s
After sacriticing the animals by decapitation the brains were quickl,, removed The brain areas: midbrain, pons and medulla obhlngata were dissected out at 4 C. "The spinal cord was also dissected and divided into three sections: cervical, thoracic and lumbar. All samples ,.,,ere immediately frozen at - 6 0 C until the assa`, v,as carried out (no more than 18 hr later). The tissues ,,,,'ere homogenized it] butanol acid mixture and 5 HT was assayed fluorometrically according to the procedure described by Curzon & Green (19701. Stati.stical methods All values herein reported represent mean _+ SEM. The statistical significance of differences between groups were calculated by means of Student's t-Test (Snedecor & Cochran. 1967). Drug~ The following drugs were used: 5 Hydroxytryptophan propionic acid: 5 7 dihydroxylryptamine bimaleale and 5-methoxy .\'-N dimethyltryptamine {Sigma): MK 486 [Merck. Sharp & Dohme): pargyline chloride (Abbot): methysergide maleate (Sandoz): fenfluramine (Newark): fluoxetine hydrochloride [Lilly); burimamide 1S. K. F. Laboratories); naloxone hydrochloride (Endo): pyrilamine maleate (Rhone-Poulene): desmethylimipramine chloride (Geigy) and pimozide (Janssen). RESII.FS
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Effeet.~ on serotonin lerels and .serotonergic h)'potensire action o]5-7 D H T A selective depletion of endogenous serotonin levels was obtained by injecting intracisternally 25/~g of serotonergic neurotoxin 5-7 DHT. In the spinal cord 5-HT levels decreased 40-50% while in the brain-stem only 10°', (Table 1). Thus, this depletion would indicate a partial destruction of serotonergic terminals in the spinal cord. according to Trulson et al. (1976). To study how these reductions on the 5-HT levels of the spinal cord affect the responses to a direct 5-HT agonist, the animals were injected intracisternally 7 days before the experiment with 5-7 DHT.
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Fig. 5. Dose-response curve relating to 5 Me O-DMT effects on mean blood pressure. 5 Me O-DMT was given i.v. to either saline or 5 7 DHT intracisternally injected rats. Ordinates shows differences in mean blood pressure. abscissa : Doses of the serotonergic direct agonist in ,~g/kg. 0 : Control group (Saline solution intracisternal. 7 days beforeJ. O: Denervated animals (5 7 DHT intracisternal, 7 days beforc). *P < 0.05. Figure 5 shows that in the control group a hypotensive effect was obtained with 1 and 3 mg/kg of 5 methoxy N - N dimethyltryptamine (5 Me O - D M T ) whereas in the denervated group a shift to the left of approximately one logarithmic unit in the dose-response curve was observed.
Effects on heart rate The treatments used affected the heart rate in different ways. Doses of 10 and 30 mg/kg of 5 0 H - T P induced a slight bradycardia. 10mg/kg: lower from 413 + 22 b p m to 372 + 20 b p m ; 3 0 m g / k g from 423 + 17 to 391 +__ 13 b p m (in b o t h groups n = 4). None of the receptor blockers used affected the heart rate (Table 2A). In contrast, when fenfluramine was administered, a significant decrease in the heart rate was obtained. This action was prevented by pretreating the animals with fluoxetine or methysergide (Table 2B).
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43(1
A c ; t s l i y Jos(; RAMI'RI:Z el O/.
Table 1. Serotonin le~.els in rats inJected either with sahne solution or 5 intracisternally
DHT
5-11T lig.g Iwet. ~tl" ('ontrol
Area Midbrain Pons Medulla oblongata Cervical spinal cord Thoracic spinal cord Lumbar spinal cord
0.87 1.11 0.83 1.63 1.16 2.44
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+_ 0.03 _+ 0.05 + 0.02 _+ 0.06 _+ 0.04 _+ 0.27
0.79 0.99 0.87 0.85 0.68 1.15
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Reduct? -x - 11 ~-6 -47 - 41 -53
Per cent of the decrease in serotonin levels induced by administering 5 7 DHT I.C.M. 7 days previous. *P < 0.05; n = 4. DISCUSSION T h e s e r o t o n i n p r e c u r s o r 5 - H T P p r o d u c e d a dosed e p e n d e n t decrease in the m e a n b l o o d pressure w h e n a d m i n i s t e r e d intravenously to n o r m o t e n s i v e anesthetized rats, being the m i n i m u m effective dose of 5 mg/kg. After a d m i n i s t r a t i o n of M K 486, an inhibitor o f peripheral decarboxylase, the s h o r t lasting fall in b l o o d pressure p r o d u c e d by 5 m g / k g o f 5 - H T P was not only e n h a n c e d but also r e m a i n e d significantly lower than c o n t r o l values for m o r e than 30 min. This effect suggests an increased availability o f 5 - H T P to the CNS. This interaction, a n d the p o t e n t i a t i o n o b served in a n i m a l s p r e t r e a t e d with a M A O I , s u p p o r t s the idea that, to p r o d u c e h y p o t e n s i o n , 5 - H T P should be previously c o n v e r t e d to s e r o t o n i n in the CNS. F u r t h e r m o r e , an a d d i t i o n a l evidence a c c o u n t i n g for the s t i m u l a t i o n o f a s e r o t o n e r g i c r e c e p t o r is p r o v i d e d
by the fact that m e t h y s e r g i d e p r e v e n t e d the reduction in b l o o d pressure i n d u c e d by 5 - H T P . T h e s e results agree with those r e p o r t e d by o t h e r a u t h o r s in different species like cats IFl6rez Armijo. 1974: F r a n z et al.. 1975: Tadepalli et al., 19771 and dogs ( A n t o n a c c i o & R o b s o n , 1973, 1975). lto & S c h a n b e r g (1972) r e p o r t e d that the inhibition o f the central t r y p t o p h a n hydroxylase in rats prod u c e d an increase in b l o o d pressure c o r r e l a t e d with the s e r o t o n e r g i c depletion. After the injection of 5 - H T P , that b y p a s s e s the enzyme, the b l o o d pressure r e t u r n e d to basal values. M o r e o v e r . p r o v o k i n g brainstem lesions surgically the 5 - H T P site o f action was localized in the rostral p o r t i o n o f the medulla o b l o n gala. O n the o t h e r hand, H e n n i n g & R u b e n s o n (1971j r e p o r t e d that 5 - H T P a d m i n i s t e r e d to c o n s c i o u s rats p r o d u c e d a significant fall in b l o o d pressure that was peripheral in origin since w h e n the peripheral decar-
Table 2(A). Effects on heart rate induced by different drug administration
Pretreatmenl + M K 486 + Parg~line + Naloxone Pinlozide + Burimarnide + P,,rilanaine.
Ileart rate ibeats.:mm) 5-HTP maximal Basal value ~ effect b 359 354 382 332 364 368 36(1 384 364 368 .~47
+ 22 4_ 24 _+ 32 !. 34 _+ 17 i 26 ÷8 + 28 ! 33 _+ 19 ± 25
343 319 415 302 355 353 346 343 358 372 345
± 26 4=_8 _+ 40 ~ 23 t- 5 _+ 5 j 9 ~ 36 .l 33 -.- 20 _t _:,~-
n 3 3 3 5 5 4 4 5 5 3 3
Table 2(B).
Pretreatment -Fluoxetine -Methysergide
Heart rate (beats.min) Fenfluramine maximal Basal value s effect h 392 _+ 14 390 + 8 365 + 15
329 ! 18" 368 + 29 345 _+ 34
n 7 5 4
Shown are the maximal effects induced by (A) 5-HTP (5 mg:kgk or (B} fenfluramine (5 mg:kg) given to animals that did not receive, or received different pretreatrnents. J Pretreatments effect on basal heart rate; and t, on either the 5-1tTP or fenfluramine maximal effect. *P < 0.05.
Serotonin on blood pressure boxylase was inhibited by pretreating the animals with MK 486, neither hypotensive effect nor 5-HT increased levels in CNS were observed. In a similar way, when the animals were pretreated with nialamide, a MAOI. and MK 486 once again no significant action of 5-HTP was obtained. However, according to Fuxe et al. (1971) the ability of the 5-HT neurons to take up and accumulate 5-HT in the presence of 5-HTP is low in spite of the large amounts of 5-HTP present in the brain after extracerebral decarboxylase inhibition: the specificity for the 5-HT neurons is obtained with 20-100mg/kg of 5-HTP. Thus, the discrepancies in Henning & Rubenson results could be due to the fact that the authors mentioned above used higher 5-HTP doses than us. Moreover, Horita & Hamilton [1970) studied the central hyperthermic action induced by 5-HTP in rabbits; doses from 35 50 mg/kg of 5-HTP produced a dose-dependent hypcrthermia. Brain 5-HT levels raised 57°~, when 35 mg/kg (i.v.t of 5-HTP was injected. In animals pretreated with the peripheral decarboxylase inhibitor, 35 mg/kg of 5-HTP, produced marked increase in temperature and a 194"~o augment in the brain 5-HT concentration. These data further support the idea that the higher doses of 5-HTP (200 mg/kg i.v.) used by Henning & Rubenson (1971 ) masked their results. The possibility that the hypotensive action could be mediated by displacement of neurotransmitters other than serotonin is open, since 5-HTP could be decarboxylated in sites other than serotonergic neurons like catecholaminergic neves or vascular endothelium (Fuxe et al., 1971; Pletscher & Bartholini, 1971; Yunger & Harvey, 19761. However, this possibility is discarded since the blockadc of serotonergic receptors by methysergide antagonized the effect of 5-HTP. Moreover, fenfluramine, a serotonergic releasing drug, also produced hypotension and this effect was antagonized by either fluoxetine or methysergide. All these results indicate that the decrease in the mean blood pressure observed after injecting 5-HTP in anesthetized rats is due to its conversion to serotonin in the CNS. Our results blocking morphine, dopamine or histamine receptors show that, the hypotension induced by 5-HTP is due to the stimulation of a serotonergic receptor and does not depend on the existence of a dopaminergic, histaminergic or opioid consecutive pathway. Previous published results (Ito & Schanberg, 1972: Tadepalli et ul., 19771 suggested that the hypotension obtained after serotonergic stimulation is mediated by a bulbo-spinal serotonergic pathway whose origin is localized in the medulla oblongata. This serotonergic action would be mediated by inhibition of preganglionic sympathetic activity. Additionally, the stimulation of mcdullar areas and ~rotonergic descending tibers in cats induced a decrease in mean blood pressure and sympathetic preganglionic activity (Franz et al., 1975). On the other hand, several reports suggested that the hypothalamic serotonergie stimulation in rats observed when administering 5-HT i.c.v. (Nahmod et al., 1978) or the stimulation of the serotonergic mesencephalic nucleus (Smits et al., 1978: Kuhn et al., 1980)
431
induced a hypertensive effect. However, in spite of increasing the brain 5-HT levels a 165~o (with 5-HTP i.c.v, plus a peripheral decarboxylase inhibitor) no changes in the blood pressure were observed (Lin, 1979). From these reports it could be suggested that the serotonergic system may be responsible, depending on the stimulated pathway, for a hypertensive or hypotensive response. Furthermore, Lambert et al. 0978) reported that the effect of 5-HT on blood pressure in rats appeared to be dependent on the manner in which the transmitter is introduced into the brain. Therefore, it is interesting to evaluate which is the effect of the serotonergic stimulation when spinal receptors are supersensitive. In animals pretreated with 5 7 DHT, 7 days before the experiment, a potentiation of the hypotensive effect of 5 Me O DMT was observed. This potentiation indicates the existence of denervation supersensitivity following destruction of bulbo-spinal serotonergic terminals and supportes the idea that these receptors, responsible for the hypotensire action, are localized in the spinal cord. These results could be an evidence supporting Franz et al. 11975) who suggested that 5-HT and noradrenaline descending pathways modulate the preganglionic sympathetic activity. Kuhn et al. (1980) reported a selective pressor effect on the ascending 5-HT neuronal system in rats, by electrical stimulation of the dorsal and median raphe nucleus, which was abolished by rostral transections of the raphe nuclei presumably by interrupting the ascending tract of the 5-HT neuronal system. On the other hand, lto & Schanberg [1972) described a bulbo-protuberancial area which could be responsible for the hypotensive effect observed after 5-HTP administration in tryptophan hydroxylase inhibited rats. Both reports further suggest that in rats 5-HT may participate in pressor and depressor areas involved in the cardiovascular response. In our case, a direct 5-HT agonist (5 Me ODMT) administered to anesthetized rats, induced a lowering of the blood pressure. Therefore, only the hypotensive descending 5-HT neuronal system as being stimulated or expressed. Our results failed to demonstrate any effect of 5-HTP on heart rate. Other authors, however, have shown that this serotonergic precursor produces bradycardia (Antonaccio & Robson, 1973, 1975; FI6rez & Armijo, 1974; Tadepalli et al., 1977). The reasons for these discrepancies are not known. Nevertheless, our results are not in complete contradiction with those of the authors cited above since fenfluramine reduced the heart rate. This effect was mediated by serotonergic stimulation since it was prevented by a 5-HT antagonist or a serotonin uptake blocker. In conclusion, our studies have shown that the increase in the serotonergic activity by injection of: a precursor or an agonist of 5-HT or a serotonergic releasing drug, reduced the blood pressure in the anesthetized rat. This effect could be due to an activation of serotonergic receptors localized in the spinal cord.
A c k n o w l e d . q e m e n t s - This work was supported by a grant from the Consejo Naeional de investigaciones Cientificas y T6cnicas [CONICET).
432
AG~.:ST/N J(>sI~ RAMiRI!Z eta/.
f h e authors wish to thank l)r Francisco J. li. Stefano for reading the manuscript and h~r his helpful criticism of the work. We also greatfully acknowledge Miss Susy Ingala for her technical assistance.
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