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Serotonergic involvement in the cardiovascular stimulation by thyrotropin-releasing hormone (TRH) in anesthetized rats
147
Neuroscience Letters, 127 (1991) 147 149 © 1991 Elsevier Scientific Publishers Ireland Ltd. 0304-3940/91/$ 03.50 ADONIS 030439409100301Q NSL 07814
Serotonergic involvement in the cardiovascular stimulation by thyrotropin-releasing hormone (TRH) in anesthetized rats Marja-Leena Nurminen 1, Ilari Paakkari 1 and Timo Sepp/il/i 2 1Department of Pharmacology and Toxicology, University of Helsinki, Helsinki (Finland) and eNational Public Health Institute, Helsinki (Finland) (Received 7 February 1991; Revised version received 21 March 1991; Accepted 22 March 199 l)
Key words: Thyrotropin-releasing hormone (TRH); Cardiovascular; Serotonergic mechanisms; 5-Hydroxytryptamine; Blood pressure; Heart rate; p-Chloroamphetamine; p-Chlorophenylalanine The cardiovascular effects of intracerebroventricularly administered thyrotropin-releasing hormone (TRH) were studied in anesthetized rats in the presence of serotonin (5-HT) depletion induced by pretreatments with p-chloroamphetamine (PCA) or p-chlorophenylalanine (PCPA). After PCA the reduction of the whole brain 5-HT and 5-HIAA (5-hydroxy-indoleacetic acid) was 53% and 32% of control, respectively. PCPA reduced the brain 5-HT and 5-HIAA levels even to a greater extent, corresponding levels were 9% and 17% of control. TRH 1-100 nmol/kg increased dose dependently blood pressure and heart rate. PCPA pretreatment significantly attenuated the pressor effect and the tachycardia induced by TRH, whereas PCA did not modify the effects of TRH, which may be related to its weaker capacity to deplete 5-HT in TRH sensitive brain areas. These results suggest the involvement of the central serotonergic system in the TRH-induced cardiovascular stimulation.
In addition to its well known endocrine actions, thyrotropin-releasing hormone (TRH) exerts a wide spectrum of central effects including those on the autonomic nervous system [19]. Administration of TRH intracerebroventricularly (i.c.v.) or directly into the brain nuclei produced a strong cardiovascular stimulation in both anesthetized and conscious animals [7, 16]. In the central nervous system endogenous TRH has been reported to co-exist in neurons containing serotonin (5-HT) [1, 11]. Biochemical studies have rendered further evidence on the co-existence of TRH and 5-HT in the nerve terminals [15]. Furthermore, a growing body of evidence supports functional associations between TRH and 5-HT, for example in hyperactivity behavior [8], sexual behavior [9] and in the respiratory effects [5]. 5-HT and TRH have also been reported to have an interaction in motoneuron control [2, 6]. In addition, inhibition of endogenous TRH with i.c.v, infusion of specific TRH antiserum increased sensitivity to both TRH and 5-HT agonists [3]. TRH and 5-HT may also interact in the control of the cardiovascular system. Serotonergic involvement in the cardiovascular and sympathoexcitatory responses to TRH has recently been demonstrated using local microinjections of TRH into serotonergic raphe nuclei as Correspondence: M.-L. Nurminen, Department of Pharmacology and Toxicology, University of Helsinki, Siltavuorenpenger 10, SF-00170 Helsinki, Finland.
well as electrolytic and chemical lesions of serotonergic structures of the brain [16]. In view of the possible interactions between central 5-HT and TRH systems, we wanted to study the cardiovascular effects of TRH using p-chloroamphetamine (PCA) [18] and p-chlorophenylalanine (PCPA) [12] as selective depletors of brain 5-HT. The experiments were performed with male Wistar rats (210-380 g) pretreated 2 weeks before with intraperitoneal (i.p.) injection of 10 mg/kg PCA (DL-p-chloroamphetamine hydrochloride, Aldrich, Germany) or 3 days before with 300 mg/kg PCPA (DL-p-chlorophenylalanine ethyl ester hydrochloride, Sigma Chemical Co., St. Louis, MO, USA), control animals received i.p. injections of physiological saline. At the day of the experiments rats were anesthetized with urethane (1.5 g/kg i.p.) for the cannulation of left femoral artery for the recording of mean arterial pressure and heart rate. I.c.v. injections of TRH (Sigma) were given using a stereotaxic device, injection volume was 10/tl. After the experiments rats were sacrificed by draining the blood from jugular vessels. For the high performance liquid chromatographic (HPLC) determination of brain 5-HT and 5hydroxy-indoleacetic acid (5-HIAA) the whole brains were removed and stored at - 7 0 ° C until analyzed. Brains (1.6-2.0 g) were weighed while still frozen and homogenized with ground glass in 5 ml of 0.5 M perchloric acid and homovanillylalcohol as an interval standard. Analysis of 5-HT and 5-HIAA was based on
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Fig. 1. Effects of various doses of intracerebroventracular TRH on mean arterial pressure (MAP, a) and heart rate (HR, b) in saline- (10 ml/kg i.p.), PCA- (10 mg/kg i,p.) and PCPA- (300 mg/kg i.p.) pretreated rats. Each point represents the mean+S.E.M, of 9-10 rats. Basal values prior to first TRH administration are shown after the symbols of the pretreatments. *P<0.05, "P<0.01, *'*P<0.001 vs saline-treated group, Dunnett's t-test.
the electrochemical detection method of Mefford [17]. However, we used Lichrosorb RP-18 column (17 #m, 4 x 125 mm) and coulometric detection with dual electrode design (ESA Coulochem Model 5100 A). The eluates were oxidated first by the upstream electrode (cell 5011) at +0.7 V and then detected by reduction at the second electrode (cell 5021) at - 0 . 2 5 V. Injection volume was 50 #1. The intra-day precision expressed as CV % was 3.1 and 15.2 for 5-HT (82 ng/ml) and 5-HIAA (140 ng/ml), respectively (n = 8). Fig. la,b summarizes the effects of PCA and PCPA on the cardiovascular effects of TRH. All results are expressed as mean ___S.E.M. ANOVA for repeated measures showed significant changes within the groups, P<0.0001 for both the mean arterial pressure and the heart rate, the differences between the groups were also significant, P < 0.0003 and P < 0.01 for the mean arterial pressure and the heart rate, respectively. Pairwise com-
parisons were performed with Dunnett's test using the saline-treated group as control. Baseline blood pressure prior to the first T R H administration did not differ between the groups, but basal heart rate was significantly higher in rats pretreated with PCPA. In salinepretreated rats, T R H 1-100 nmol/kg administered i.c.v. increased mean arterial pressure and heart rate dose dependently. PCA did not alter the cardiovascular responses to T R H while PCPA significantly reduced the pressor effect (Fig. l a). Also the tachycardia produced by T R H was reduced after PCPA, but this effect may be related to the higher baseline level of H R in this group (Fig. lb). Brain 5-HT and 5 - H I A A levels in P C A - and P C P A treated rats were c o m p a r e d with those from saline treated animals (Fig. 2). Both P C A and P C P A reduced significantly the whole brain 5-HT and 5 - H I A A . After P C A the content o f brain 5-HT was 53% o f control, while after P C P A the 5-HT content o f brain was reduced to negligible levels (9% o f control). The levels o f the 5H T main metabolite 5 - H I A A were 32% and 17% o f control levels after P C A and P C P A , respectively. Both P C A and P C P A produced a substantial depletion o f the whole brain 5-HT. However, there is a marked difference between these two drugs in the regional pattern o f the reduction in brain 5-HT. Morphological changes after P C A were primarily located in serotonergic cell g r o u p in the mesencephalic ventral midbrain tegm e n t u m indicating d a m a g e in a b o u t 80% o f cells, whereas only 10% o f cells within dorsal and medial raphe nuclei showed neurotoxic signs after P C A [10]. Furthermore, the concentration o f 5-HT after P C A was not decreased in dorsal and medial raphe nuclei although a significant reduction was observed in ventral midbrain tegmentum [4]. Since serotonergic mechanisms located in the dorsal raphe nucleus were suggested to be involved
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Fig. 2. Effects of pretreatments with saline (10 ml/kg i.p.), PCA (10 mg/ kg i.p.) or PCPA (300 mg/kg i.p.) on the whole brain 5-HT and 5HIAA contents. Each bar represents the mean + S.E.M. of 9-10 experiments. *'P < 0.01, ""P < 0.001 vs saline-treated group, Dunnett's t-test.
149 in the c a r d i o v a s c u l a r effects o f T R H [16], the i n a b i l i t y o f P C A to deplete 5 - H T in this nucleus m a y p r o v i d e an e x p l a n a t i o n for the failure o f P C A to p r o d u c e altera t i o n s in the c a r d i o v a s c u l a r effects o f T R H . P C P A c a u s e d a n e a r l y c o m p l e t e d e p l e t i o n o f 5 - H T in all a r e a s o f the b r a i n [12]. In o u r s t u d y P C P A p r o d u c e d a greate r t h a n 90% decrease in whole b r a i n 5 - H T levels, thus it is likely t h a t this r e d u c t i o n o c c u r r e d also in s e r o t o n e r g i c structures sensitive to the c a r d i o v a s c u l a r s t i m u l a t i o n b y T R H . P C P A is k n o w n to deplete also p e r i p h e r a l 5 - H T storages [12], b u t since the c a r d i o v a s c u l a r effects o f T R H are p r e d o m i n a n t l y centrally m e d i a t e d [19], p e r i p h e r a l int e r a c t i o n with T R H a n d 5 - H T seems unlikely. T h e r e is p h a r m a c o l o g i c a l evidence t h a t b o t h 5 - H T a n d T R H m a y be i n v o l v e d in the c a r d i o v a s c u l a r regulation. I.c.v. injections o f small a m o u n t s o f 5 - H T increased the b l o o d pressure in rats [13, 14]. O u r results s h o w t h a t after m a r k e d d e p l e t i o n o f b r a i n 5 - H T the T R H - i n d u c e d p r e s s o r a n d t a c h y c a r d i c effects were a t t e n u a t e d suggesting t h a t T R H interacts with 5 - H T in the c a r d i o v a s c u l a r regulation. T h e a u t h o r s wish to express their g r a t i t u d e to Mrs. V u o k k o Pahlsten, Mrs. Beate Sarell a n d M r . K a r i A r i niemi for their skillful technical assistance, a n d to P r o fessor M.J. M a t t i l a , M . D . , for his s u p p o r t a n d readiness to give advice. F i n a n c i a l s u p p o r t was p r o v i d e d by the P h a r m a c a l R e s e a r c h F o u n d a t i o n a n d the R e s e a r c h C o u n c i l o f M e d i c i n e , the A c a d e m y o f F i n l a n d .
1 Appel, N.M., Wessendorf, M.W. and Elde, R.P., Thyrotropinreleasing hormone in spinal cord: coexistence with serotonin and with substance P in fibers and terminals apposing identified preganglionic sympathetic neurons, Brain Res., 415 (1987) 137-143. 2 Barbeau, H. and Bedard, P., Similar motor effects of 5-HT and TRH in rats following chronic spinal transection and 5,7-dihydroxytryptamine injection, Neuropharmacology, 20 (1981) 477-481. 3 Bennett, G.W., Edwards R.M., Lighton, C. and Marsden, C.A., Thyrotrophin releasing hormone - - 5-hydroxytryptamine interactions in the brain studied using chronic immunization and chemical lesioning techniques, J. Recept. Res., 7 (1987) 555-579. 4 Bertilsson, L., Koslow, S.H. and Costa, E., 5-Hydroxytryptamine depletion in mesencephalic nuclei of rat brain following a single injection ofp-chloroamphetamine, Brain Res., 91 (1975) 348-350. 5 Carter, D.A. and Lightman, S.L., Cardio-respiratory actions of
substance P, TRH and 5-HT in the nucleus tractus solitarius of rats: evidence for functional interactions of neuropeptides and amine neurotransmitters, Neuropeptides, 6 (1985) 425-436. 6 Clarke, K.A., Parker, A.J. and Stirk, G.C., Potentiation of motoneurone excitability by combined administration of 5-HT agonist and TRH analogue, Neuropeptides, 6 (1985) 269-282. 7 Feuerstein, G., Hassen, A.H. and Faden, A.I., TRH: cardiovascular and sympathetic modulation in brain nuclei of the rat, Peptides, 4 (1983) 617-620. 8 Green, A.R. and Grahame-Smith, D.G., TRH potentiates behavioral changes following increased brain 5-hydroxytryptamine accumulations in rats, Nature, 251 (1974) 524-526. 9 Hansen, S., Svensson, L., Hrkfelt, T. and Everitt, B.J., 5-hydroxytryptamine - - thyrotropin releasing hormone interactions in the spinal cord: effects on parameters of sexual behaviour in the male rat, Neurosci. Lett., 42 (1983) 299-304. 10 Harvey, J.A., McMaster, S.E. and Yunger, L.M., p-Chloroamphetamine: selective neurotoxic action in brain, Science, 187 (1975) 841-843. 11 Johansson, O., Hrkfelt, T., Pernow, B., Jeffcoate, S.L., White, N., Steinbusch, H.W.M., Verhofstad, A.A.J., Emson, P.C. and Spindel, E., Immunohistochemical support for three putative transmitters in one neuron: coexistence of 5-hydroxytryptamine, substance P- and thyrotropin releasing hormone-like immunoreactivity in medullary neurons projecting to the spinal cord, Neuroscience, 6 (1981) 1857-1881. 12 Koe, B.K. and Weissman, A., p-Chlorophenylalanine: a specific depictor of brain serotonin, J. Pharmacol. Exp. Ther., 154 (1966) 499-516. 13 Kristic, M.K. and Djurkovic, D., Analysis of cardiovascular responses to central administration of 5-hydroxytryptamine in rats, Neuropharmacology, 19 (1980) 455-463. 14 Lambert, G., Friedman, E. and Gershon, S., Centrally-mediated cardiovascular responses to serotonin, Life Sci., 17 (1975) 915-920. 15 Marsden, C.A., Bennett, G.W., Irons, J., Gilbert, R.F.T. and Emson, P.C., Localization and release of 5-hydroxy-tryptamine, thyrotrophin releasing hormone and substance P in rat ventral spinal cord, Comp. Biochem. Physiol., 72C (1982) 263-270. 16 Mattila, J. and Bunag, R.D., Pressor and sympathetic responses to dorsal raphe nucleus infusions of TRH in rats, Am. J. Physiol,, 258 (1990) R1464-R1471. 17 Mefford, I.N., Application of high performance liquid chromatography with electrochemical detection to neurochemical analysis: measurement of catecholamines, serotonin and metabolites in rat brain, J. Neurosci. Meth., 3 (1981) 207-224. 18 Sanders-Bush, E., Bushing, J.A. and Sulser, F., Long term effects ofp-chloroamphetamine and related drugs on central serotonergic mechanisms, J. Pharmacol. Exp. Ther., 192 (1975) 33-41. 19 Siren, A.-L. and Feuerstein, G., Central autonomic pharmacology of thyrotropin releasing hormone, Rev. Clin. Basic Pharmacol., 6 (1987) 315-327.
Neuroscience Letters, 127 (1991) 147 149 © 1991 Elsevier Scientific Publishers Ireland Ltd. 0304-3940/91/$ 03.50 ADONIS 030439409100301Q NSL 07814
Serotonergic involvement in the cardiovascular stimulation by thyrotropin-releasing hormone (TRH) in anesthetized rats Marja-Leena Nurminen 1, Ilari Paakkari 1 and Timo Sepp/il/i 2 1Department of Pharmacology and Toxicology, University of Helsinki, Helsinki (Finland) and eNational Public Health Institute, Helsinki (Finland) (Received 7 February 1991; Revised version received 21 March 1991; Accepted 22 March 199 l)
Key words: Thyrotropin-releasing hormone (TRH); Cardiovascular; Serotonergic mechanisms; 5-Hydroxytryptamine; Blood pressure; Heart rate; p-Chloroamphetamine; p-Chlorophenylalanine The cardiovascular effects of intracerebroventricularly administered thyrotropin-releasing hormone (TRH) were studied in anesthetized rats in the presence of serotonin (5-HT) depletion induced by pretreatments with p-chloroamphetamine (PCA) or p-chlorophenylalanine (PCPA). After PCA the reduction of the whole brain 5-HT and 5-HIAA (5-hydroxy-indoleacetic acid) was 53% and 32% of control, respectively. PCPA reduced the brain 5-HT and 5-HIAA levels even to a greater extent, corresponding levels were 9% and 17% of control. TRH 1-100 nmol/kg increased dose dependently blood pressure and heart rate. PCPA pretreatment significantly attenuated the pressor effect and the tachycardia induced by TRH, whereas PCA did not modify the effects of TRH, which may be related to its weaker capacity to deplete 5-HT in TRH sensitive brain areas. These results suggest the involvement of the central serotonergic system in the TRH-induced cardiovascular stimulation.
In addition to its well known endocrine actions, thyrotropin-releasing hormone (TRH) exerts a wide spectrum of central effects including those on the autonomic nervous system [19]. Administration of TRH intracerebroventricularly (i.c.v.) or directly into the brain nuclei produced a strong cardiovascular stimulation in both anesthetized and conscious animals [7, 16]. In the central nervous system endogenous TRH has been reported to co-exist in neurons containing serotonin (5-HT) [1, 11]. Biochemical studies have rendered further evidence on the co-existence of TRH and 5-HT in the nerve terminals [15]. Furthermore, a growing body of evidence supports functional associations between TRH and 5-HT, for example in hyperactivity behavior [8], sexual behavior [9] and in the respiratory effects [5]. 5-HT and TRH have also been reported to have an interaction in motoneuron control [2, 6]. In addition, inhibition of endogenous TRH with i.c.v, infusion of specific TRH antiserum increased sensitivity to both TRH and 5-HT agonists [3]. TRH and 5-HT may also interact in the control of the cardiovascular system. Serotonergic involvement in the cardiovascular and sympathoexcitatory responses to TRH has recently been demonstrated using local microinjections of TRH into serotonergic raphe nuclei as Correspondence: M.-L. Nurminen, Department of Pharmacology and Toxicology, University of Helsinki, Siltavuorenpenger 10, SF-00170 Helsinki, Finland.
well as electrolytic and chemical lesions of serotonergic structures of the brain [16]. In view of the possible interactions between central 5-HT and TRH systems, we wanted to study the cardiovascular effects of TRH using p-chloroamphetamine (PCA) [18] and p-chlorophenylalanine (PCPA) [12] as selective depletors of brain 5-HT. The experiments were performed with male Wistar rats (210-380 g) pretreated 2 weeks before with intraperitoneal (i.p.) injection of 10 mg/kg PCA (DL-p-chloroamphetamine hydrochloride, Aldrich, Germany) or 3 days before with 300 mg/kg PCPA (DL-p-chlorophenylalanine ethyl ester hydrochloride, Sigma Chemical Co., St. Louis, MO, USA), control animals received i.p. injections of physiological saline. At the day of the experiments rats were anesthetized with urethane (1.5 g/kg i.p.) for the cannulation of left femoral artery for the recording of mean arterial pressure and heart rate. I.c.v. injections of TRH (Sigma) were given using a stereotaxic device, injection volume was 10/tl. After the experiments rats were sacrificed by draining the blood from jugular vessels. For the high performance liquid chromatographic (HPLC) determination of brain 5-HT and 5hydroxy-indoleacetic acid (5-HIAA) the whole brains were removed and stored at - 7 0 ° C until analyzed. Brains (1.6-2.0 g) were weighed while still frozen and homogenized with ground glass in 5 ml of 0.5 M perchloric acid and homovanillylalcohol as an interval standard. Analysis of 5-HT and 5-HIAA was based on
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Fig. 1. Effects of various doses of intracerebroventracular TRH on mean arterial pressure (MAP, a) and heart rate (HR, b) in saline- (10 ml/kg i.p.), PCA- (10 mg/kg i,p.) and PCPA- (300 mg/kg i.p.) pretreated rats. Each point represents the mean+S.E.M, of 9-10 rats. Basal values prior to first TRH administration are shown after the symbols of the pretreatments. *P<0.05, "P<0.01, *'*P<0.001 vs saline-treated group, Dunnett's t-test.
the electrochemical detection method of Mefford [17]. However, we used Lichrosorb RP-18 column (17 #m, 4 x 125 mm) and coulometric detection with dual electrode design (ESA Coulochem Model 5100 A). The eluates were oxidated first by the upstream electrode (cell 5011) at +0.7 V and then detected by reduction at the second electrode (cell 5021) at - 0 . 2 5 V. Injection volume was 50 #1. The intra-day precision expressed as CV % was 3.1 and 15.2 for 5-HT (82 ng/ml) and 5-HIAA (140 ng/ml), respectively (n = 8). Fig. la,b summarizes the effects of PCA and PCPA on the cardiovascular effects of TRH. All results are expressed as mean ___S.E.M. ANOVA for repeated measures showed significant changes within the groups, P<0.0001 for both the mean arterial pressure and the heart rate, the differences between the groups were also significant, P < 0.0003 and P < 0.01 for the mean arterial pressure and the heart rate, respectively. Pairwise com-
parisons were performed with Dunnett's test using the saline-treated group as control. Baseline blood pressure prior to the first T R H administration did not differ between the groups, but basal heart rate was significantly higher in rats pretreated with PCPA. In salinepretreated rats, T R H 1-100 nmol/kg administered i.c.v. increased mean arterial pressure and heart rate dose dependently. PCA did not alter the cardiovascular responses to T R H while PCPA significantly reduced the pressor effect (Fig. l a). Also the tachycardia produced by T R H was reduced after PCPA, but this effect may be related to the higher baseline level of H R in this group (Fig. lb). Brain 5-HT and 5 - H I A A levels in P C A - and P C P A treated rats were c o m p a r e d with those from saline treated animals (Fig. 2). Both P C A and P C P A reduced significantly the whole brain 5-HT and 5 - H I A A . After P C A the content o f brain 5-HT was 53% o f control, while after P C P A the 5-HT content o f brain was reduced to negligible levels (9% o f control). The levels o f the 5H T main metabolite 5 - H I A A were 32% and 17% o f control levels after P C A and P C P A , respectively. Both P C A and P C P A produced a substantial depletion o f the whole brain 5-HT. However, there is a marked difference between these two drugs in the regional pattern o f the reduction in brain 5-HT. Morphological changes after P C A were primarily located in serotonergic cell g r o u p in the mesencephalic ventral midbrain tegm e n t u m indicating d a m a g e in a b o u t 80% o f cells, whereas only 10% o f cells within dorsal and medial raphe nuclei showed neurotoxic signs after P C A [10]. Furthermore, the concentration o f 5-HT after P C A was not decreased in dorsal and medial raphe nuclei although a significant reduction was observed in ventral midbrain tegmentum [4]. Since serotonergic mechanisms located in the dorsal raphe nucleus were suggested to be involved
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BRAIN 5 - H I A A
Fig. 2. Effects of pretreatments with saline (10 ml/kg i.p.), PCA (10 mg/ kg i.p.) or PCPA (300 mg/kg i.p.) on the whole brain 5-HT and 5HIAA contents. Each bar represents the mean + S.E.M. of 9-10 experiments. *'P < 0.01, ""P < 0.001 vs saline-treated group, Dunnett's t-test.
149 in the c a r d i o v a s c u l a r effects o f T R H [16], the i n a b i l i t y o f P C A to deplete 5 - H T in this nucleus m a y p r o v i d e an e x p l a n a t i o n for the failure o f P C A to p r o d u c e altera t i o n s in the c a r d i o v a s c u l a r effects o f T R H . P C P A c a u s e d a n e a r l y c o m p l e t e d e p l e t i o n o f 5 - H T in all a r e a s o f the b r a i n [12]. In o u r s t u d y P C P A p r o d u c e d a greate r t h a n 90% decrease in whole b r a i n 5 - H T levels, thus it is likely t h a t this r e d u c t i o n o c c u r r e d also in s e r o t o n e r g i c structures sensitive to the c a r d i o v a s c u l a r s t i m u l a t i o n b y T R H . P C P A is k n o w n to deplete also p e r i p h e r a l 5 - H T storages [12], b u t since the c a r d i o v a s c u l a r effects o f T R H are p r e d o m i n a n t l y centrally m e d i a t e d [19], p e r i p h e r a l int e r a c t i o n with T R H a n d 5 - H T seems unlikely. T h e r e is p h a r m a c o l o g i c a l evidence t h a t b o t h 5 - H T a n d T R H m a y be i n v o l v e d in the c a r d i o v a s c u l a r regulation. I.c.v. injections o f small a m o u n t s o f 5 - H T increased the b l o o d pressure in rats [13, 14]. O u r results s h o w t h a t after m a r k e d d e p l e t i o n o f b r a i n 5 - H T the T R H - i n d u c e d p r e s s o r a n d t a c h y c a r d i c effects were a t t e n u a t e d suggesting t h a t T R H interacts with 5 - H T in the c a r d i o v a s c u l a r regulation. T h e a u t h o r s wish to express their g r a t i t u d e to Mrs. V u o k k o Pahlsten, Mrs. Beate Sarell a n d M r . K a r i A r i niemi for their skillful technical assistance, a n d to P r o fessor M.J. M a t t i l a , M . D . , for his s u p p o r t a n d readiness to give advice. F i n a n c i a l s u p p o r t was p r o v i d e d by the P h a r m a c a l R e s e a r c h F o u n d a t i o n a n d the R e s e a r c h C o u n c i l o f M e d i c i n e , the A c a d e m y o f F i n l a n d .
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