Brain Research, 526 (1990) 313-317 Elsevier

313

BRES 24251

Evidence that endogenous catecholamines are involved in a2-adrenoceptor-mediated modulation of the aortic baroreceptor reflex in the nucleus tractus solitarii of the rat Takao Kubo, Yoshio Goshima, Hiroyoshi Hata* and Yoshimi Misu Department of Pharmacology, Yokohama City University School of Medicine, Yokohama (Japan) (Accepted 15 May 1990) Key words: Catecholamine; Nucleus tractus solitarii; Tyramine; Yohimbine; Aortic nerve; Blood pressure; Rat

Microinjections of a-methylnoradrenaline and tyramine into the rat nucleus tractus solitarii (NTS) potentiated the depressor and bradycardic responses to aortic nerve stimulation whereas yohimbine injected similarly inhibited them. NTS pretreatment with yohimbine inhibited the baroreflex potentiation effects of a-methylnoradrenaline and tyramine whereas intraventricular pretreatment with 6-hydroxydopamine inhibited only that of tyramine. These results provide evidence that endogenous catecholamines in the rat NTS are involved in alpha2 adrenoceptor-mediated modulation of the aortic baroreceptor reflex.

The nucleus tractus solitarii (NTS), the principal site of termination of the primary afferent fibers from peripheral baroreceptors, is densely innervated by catecholaminergic neurons 4'7'17 and contains a high density of a 2adrenoceptors 26. From many studies 14'15'24'28, catecholamines and a2-adrenoceptors in the NTS have been implicated in the central cardiovascular control. One role that has been suggested for catecholamines in the NTS is in the modulation of baroreceptor reflexes 5' 19,23 In the present study, we examined effects of a-methylnoradrenaline, an a-adrenergic agonist, yohimbine, an a-adrenergic antagonist, and tyramine, a catecholamine-releasing agent, injected into the rat NTS, on blood pressure and heart rate responses to aortic nerve stimulation, the objective being to obtain evidence for a baroreflex modulation role of endogenous catecholamines in the NTS. Studies were performed on male Wistar rats (310-350 g) anesthetized with urethane (1.2 g/kg, i.p.). The femoral artery was cannulated to enable blood pressure to be continuously recorded. The rats were paralyzed with D-tubocurarine (1 mg/kg, i.m.) and artificially ventilated with a respirator 1°. Tidal volumes were chosen according to the ventilation standards for small mammals described by Kleinman and Radford 8 and end-tidal p C O 2 levels were monitored using a clinical gas monitor (Sanei 1H26). The animal was placed in a stereotaxic apparatus

with the head fixed at 45 ° . The dorsal surface of the lower brainstem was exposed by a limited occipital craniotomy. Microinjections were made unilaterally into the left NTS at a level 0.6 m m rostral to the caudal tip of the area postrema, a-Methylnoradrenaline and tyramine hydrochloride dissolved in saline (0.9% NaCl in phosphate buffer, p H 7.4) were given in a volume of 30 nl in 2 s through a glass micropipette (tip diameter 40-60 p m ) connected to a 1-/,1 Hamilton microsyringe and a micrometer as described 12. Yohimbine hydrochloride was microinjected in a volume of 100 nl in 20 s for its low solubility. The injection site was marked at the end of some experiments by injecting 30 nl of concentrated solution of Evans blue dye. The brain was removed and frozen sections were cut (50 p m ) , and the injection site was identified 1°. The left aortic nerve in the neck was located with the aid of an operation microscope (Olympus, X-2) as described 18. The central end of the nerve was placed on bipolar platinum electrodes and stimulated every 2-3 min with rectangular pulses at 3-10 V, 0.1 ms and 20 Hz for 10 S 12. In some experiments, for destruction of central catecholaminergic fibers rats were given two injections of 6-hydroxydopamine hydrobromide (6-OHDA, 250/zg) or vehicle (0.5% ascorbic acid in saline) into the lateral brain ventricle at 3-day intervals 1 week before the

* Present address: Pharmacological Division, Research Laboratories, Nippon Shoji, Ltd., Ibaraki, Osaka 567, Japan. Correspondence: T. Kubo, Department of Pharmacology, Yokohama City University, School of Medicine, Fukuura, Kanazawa-ku, Yokohama 236, Japan. 0006-8993/90/$03.50 © 1990 Elsevier Science Publishers B.V. (Biomedical Divisionj

314 min. Yohimbine itself caused only a small increase in heart rate (+5.1 + 1.3 beats/min, n = 6) (P < 0.05) but did not affect blood pressure. When yohimbine (0.26 nmol) was pretreated at the depressor sites in the NTS 5 min before a-methylnoradrenaline, the potentiation effect of a-methylnoradrenaline (0.46 nmol) on the baroreflex responses was markedly reduced (Fig. 2A). Microinjections of tyramine (3.5 nmol) into the medial area of the NTS also potentiated the depressor and bradycardic responses to aortic nerve stimulation (Figs. 1C and 2A). The potentiation effect of tyramine lasted for 12-18 min. Tyramine itself decreased blood pressure by 7.4 + 1.4 mm Hg (n = 7, P < 0.05) in 4-6 min after injection. When yohimbine (0.26 nmol) was pretreated at the depressor sites in the NTS 5 min before tyramine, the potentiation effect of tyramine (3.5 nmol) on the baroreflex responses was markedly reduced (Fig. 2A). Postmortem histological examination confirmed that the injection sites were always located in the medial area of the NTS. Intraventricular pretreatment with 6-OHDA (250/~g x 2) abolished the potentiation effect of tyramine (3.5 nmol) on the aortic baroreflex responses in 6 rats whereas in the same rats the baroreflex potentiation effect of a-methylnoradrenaline (0.46 nmol) was still observed (Fig. 2B). On the other hand, in another 6 rats pretreated with intraventricular injection of vehicle, both adrenergic agonists caused a potentiation of the baroreflex responses (Fig. 2B). In the 6-OHDA-treated rats as compared with the vehicle-treated rats, 70 and 24% reductions were seen in medulla oblongata noradrenaline and adrenaline, respectively, while no significant change was observed in medulla oblongata dopamine (Table I). In the present study, microinjections of the alpha

experiments, using a stereotaxic technique]L After termination of the experiments, catecholamine contents in the medulla oblongata were estimated, using a highperformance liquid chromatography with an electrochemical detector 25. Results are reported as mean _+ S.E.M. Statistical evaluations were made using Student's t-test or paired t-test. The mean arterial pressure was 92 _+ 2 mm Hg (n = 39) and heart rate 384 + 9 beats/min (n = 39) in anesthetized rats. Stimulation of the aortic nerve (20 Hz, 10 s) produced decreases in blood pressure and heart rate (Fig. 1). We first identified depressor sites in the medial area of the NTS by injecting the excitatory amino acid L-glutamate (0.16 nmol). When a-methylnoradrenaline (0.46 nmol) was injected into the depressor sites identified with L-glutamate in the NTS, the depressor and bradycardic responses to aortic nerve stimulation were potentiated (Figs. 1A and 2A). The potentiation effect of a-methylnoradrenaline lasted 20-30 min. a-Methylnoradrenaline itself caused decreases in blood pressure and heart rate which began slowly and reached a peak (-9.6 _+ 2.4 mm Hg (n -- 8, P < 0.05) and -8.7 _+ 2.8 beats/min (n -- 8, P < 0.05), respectively) in 4-6 min after injection. In 6 other rats, a-methylnoradrenaline (0.46 nmol) injected into the NTS also potentiated the depressor response to L-glutamate (0.16 nmol) injected at the same sites in the NTS, which was -33.1 + 6.4 mm Hg 4-7 rain after a-methylnoradrenaline as compared with -18.4 _+ 4.1 mm Hg before a-methyinoradrenaline (P < 0.05). In contrast, yohimbine (0.26 nmol) injected into the area of the rat NTS inhibited the depressor and bradycardic responses to aortic nerve stimulation (Figs. 1B and 2A). The inhibition effect of yohimbine lasted for 15-20

150[

A

C 8 mln

mln

5mln

ou /.20r •

30C L

IbC 12~

f

300L

'

d-MelhyI-NA 0./.6 nmol

B

Tyramlne 3 5 nmoJ

8 rain

~o~

7~ 50 L /.20[ y

• 300 L



I

I

I

Yohcnb~ne 0.26 nmol

Fig. 1. Effects of microinjectionsof a-methylnoradrenaline (a-methyl-NA) (A), yohimbine (B) and tyramine (C) into the area of the rat NTS, on the blood pressure (BP, mm Hg) and heart rate (HR, beats/min) responses to aortic nerve stimulation (20 Hz, 10 s, squares).

315

B

.A.

Before treatment ~ After treatment VehicleJi.v t.] 6-OH-DAli.v.t.]

~ Before treatment After treatment ,

I

A f t e r yohimbine

11

30

~ o Saline a-Methyl- Tyramine Yohimbinect-Methyl- Tyramine NA 3.5 nmol 0.26nmol NA 3.5 nmol 0 Z.5 nmol 0.46nmol



,I

t-------

-iti 1 3oti -20

Tyramine ct-I~ethyl- Tyram ine o(-Methyt3.5 nmol NA 3.5 nmol NA 0Z.5 nmol 0./.6 nmol I

Treatment

i

I

Treatment

Fig. 2. A: effects of microinjections of saline, a-methylnoradrenaline (a-methyI-NA), tyramine and yohimbine into the area of the rat NTS on decreases in mean blood pressure (BP, mm Hg) and heart rate (HR, beats/min) induced by aortic nerve stimulation (20 Hz, 10 s), and effects of NTS pretreatment with yohimbine on the baroreflex modulation effects of a-methyl-NA and tyramine. The effects of drugs were determined 4-7 min after injection. Yohimbine was microinjected into the NTS 5 min before a-methyl-NA or tyramine. Values are the mean + S.E.M. from 6--8 experiments. B: effects of intraventricular (i.v.t.) pretreatment with vehicle (left 4 columns) and 6-hydroxydopamine (6-OH-DA, 250/~g x 2) (right 4 columns) on the baroreflex modulation effects of tyramine and a-methyl-NA. The effects of drugs were determined 4-7 min after injection. Values are the mean + S.E.M. from 6 experiments. *P < 0.05, compared with before treatment (paired t-test). adrenergic agonist a-methylnoradrenaline into the medial area of the rat NTS potentiated the depressor and bradycardic responses to electrical stimulation of aortic nerve afferents which contain baroreceptor afferents TM, and this potentiation effect was markedly reduced by NTS pretreatment with the a-adrenergic antagonist yohimbine, thereby suggesting the presence of a-adrenoceptors responsible for facilitatory modulation of barereceptor reflexes within the rat NTS. Since a-methylnoradrenaline and yohimbine have considerable selectivity for a2-adrenoceptors 15'2°, it appears that the NTS aadrenoceptors are of the a2-type with respect to their pharmacological characteristics.

TABLE I Effects of intraventricular injections of vehicle and 6-hydroxydopamine on endogenous catecholamine levels in rat medulla oblongata

Rats were injected with two doses of 6-OHDA (250/~g), or vehicle. Values are the mean _+S.E.M. from 6 rats.

Vehicle-treated 6-OHDA-treated

Noradrenaline (nmol/g wet wt.)

Adrenaline (pmol/g wet wt.)

Dopamine (pmol/g wet wt.)

2.85 + 0.18 0.87+ 0.03*

44.3 + 1.8 33.7 + 1.3"

219 + 13 242 + 12

*P < 0.05, compared with vehicle-treated.

The catecholamine-releasing agent tyramine injected into the NTS also enhanced the depressor and bradycardic responses to aortic nerve stimulation, which was inhibited by NTS pretreatment with yohimbine. The potentiation effect of tyramine but not of a-methylnoradrenaline was inhibited by intraventricular pretreatment with the catecholamine depleting agent 6-OHDA. These results suggest that endogenous catecholamines in the rat NTS can activate a2-adrenoceptors responsible for facilitatory modulation of baroreceptor reflexes. These findings are compatible with those of Snyder et al. 19 and Talman et al. 23 that in rats selective destruction of the catecholamine innervation of the NTS by NTS microinjection of 6 - O H D A or by destruction of A2 catecholaminergic neurons, the major source of catecholamines in the NTS 4'21, inhibits the reflex bradycardia induced by phenylephrine-induced hypertension. Since yohimbine itself injected into the rat NTS caused an inhibition of the cardiovascular responses to aortic nerve stimulation, it appears that a2-adrenoceptors in the rat NTS are tonically activated by endogenous catecholamines. a2-Adrenoceptors are known to be located both presynaptically and postsynaptically in central catecholaminergic synapses 15'26'27. In the present study, however, destruction of central catecholaminergic neurons with

316 6 - O H D A did not affect the baroreflex modulation effect of a-methylnoradrenaline, thereby suggesting that a 2adrenoceptors responsible for the baroreflex modulation are mainly located postsynaptically in the NTS catecholaminergic synapses, i.e. on cell bodies or axon terminals of non-catecholaminergic neurons. Since a2-adrenoceptors on axon terminals are known to be involved in modulation of the release of neurotransmitters from the axon terminals 3, it might be speculated that terminal a2-adrenoceptor-mediated modulation of the release of aortic baroreceptor afferent neurotransmitters in the NTS is responsible for the facilitatory modulation of the aortic baroreceptor reflex. This seems unlikely, however, since a2-adrenoceptors on axon terminals usually mediate inhibitory modulation of the release of neurotransmitters 3. In support of this idea, in the present study, a-methylnoradrenaline also potentiated the depressor response to L-glutamate which excites cell bodies of neurons but not axons of passage 6. While exact mechanisms for the a2-adrenoceptor-mediated modulation of baroreceptor reflexes remain to be settled, a recent electrophysiological study has demonstrated that in superfused rat brain slices, clonidine, another a2-adrenergic agonist, induces an increase in postsynaptic responsiveness to input from the solitary tract in NTS neurons 5. Which catecholamine(s) in the rat NTS are responsible for the modulation of the aortic baroreflex regulation can not be determined, since the rat NTS contains dopaminergic, noradrenergic and adrenergic terminals 4'7'17, microinjections of dopamine, noradrenaline and adrenaline into the rat NTS cause decreases in blood pressure and 1 Bhaskaran, D. and Freed, C.R., Changes in arterial blood pressure lead to baroreceptor-mediated changes in norepinephrine and 5-hydroxyindoleacetic acid in rat nucleus tractus solitarius, J. Pharmacol. Exp. Ther., 245 (1988) 356-363. 2 Casto, R. and Phillips, M.I., Angiotensin II attenuates baroreflexes at nucleus tractus solitarius of rats, Am. J. Physiol., 250 (1986) RI93-R198. 3 Chesselet, M.-E, Presynaptic regulation of neurotransmitter release in the brain: facts and hypotheses, Neuroscience, 12 (1984) 347-375. 4 Dahlstr6m, A. and Fuxe, K., Evidence for the existence of monoamine-containing neurons in the central nervous system. I. Demonstration of monoamines in the cell bodies of brainstem neurons, Acta Physiol. Scand., Suppl. 232 (1964) 1-55. 5 Feldman, P.D. and Feider, R.B., a2-Adrenergic modulation of synaptic excitability in the rat nucleus tractus solitarius, Brain Research, 480 (1989) 190-197. 6 Goodchild, A.K., Dampney, R.A.L. and Bandler, R.A., A method for evoking physiological responses by stimulating of cell bodies, but not axons of passage, within localized regions of the central nervous system, J. Neurosci. Methods, 6 (1982) 351-363. 7 Kalia, M., Fuxe, K. and Goldstein, M., Rat medulla oblongata. II. Dopaminergic, noradrenergic (A1 and A2) and adrenergic, neurons, nerve fibers, and presumptive terminal processes, J. Comp. Neurol., 233 (1985) 308-332. 8 Kleinman, L.I. and Radford, E.P., Ventilation standards for

heart rate 15'28, and in addition, changes in blood pressure lead to changes in the release of these three catecholamines from this region 9. From the results of the present study, however, that intraventricular pretreatment with 6 - O H D A , which inhibited the baroreflex potentiation effect of tyramine, decreased concentrations of noradrenaline and adrenaline but not dopamine in the rat medulla oblongata, it can be speculated that especially noradrenaline and/or adrenaline in the rat NTS play an important role in the baroreflex modulation. Indeed, in the rat NTS, changes in blood pressure lead to change in noradrenaline c o n t e n t and these changes in noradrenaline content are attenuated by sinoaortic denervation 1. Dopamine neurons are known to be less sensitive to the destructive effects of 6 - O H D A than noradrenaline neurons 16. It has been suggested that in the NTS, several substances including excitatory amino acids 12"22, y-aminobutyric acid 13 and some neuropeptides 2 are involved in the modulation of baroreceptor reflexes. The results of the present study provide evidence that catecholamines in the rat NTS are also involved in this modulation. In addition, since a marked enhancement and a considerable inhibition of the responses to aortic nerve stimulation were observed with a-methylnoradrenaline and yohimbine, respectively, it appears that the NTS catecholamine system may play a significant role in the central modulation of baroreceptor reflexes.

This work was supported by a Research Grant for Cardiovascular Diseases (63C-2) from the Ministry of Health and Welfare, Japan. small mammals, J. Appl. Physiol., 19 (1964) 360-362. 9 Kobilansky, C., Lanzinger, I. and Philippu, A., Release of endogenous catecholamines in the nucleus tractus solitarii during experimentally induced blood pressure changes, NaunynSchmiedeberg's Arch. Pharmacol., 337 (1988) 125-130. 10 Kubo, T. and Amano, H., Vasopressin-induced pressor responses in rats to bilateral electrolytic lesioning of the caudal portion of the nucleus tractus solitarii, Brain Research, 363 (1986) 183-187. 11 Kubo, T. and Hashimoto, M., Effects of intraventricular and intraspinal 6-hydroxydopamine on blood pressure of spontaneously hypertensive rats, Arch. Int. Pharmacodyn., 232 (1978) 166-176. 12 Kubo, T. and Kihara, M., Evidence of N-methyl-D-aspartate receptor-mediated modulation of the aortic baroreceptor reflex in the rat nucleus tractus solitarii, Neurosci. Lett., 87 (1988) 69-74. 13 Kubo, T. and Kihara, M., Evidence for v-aminobutyric acid receptor-mediated modulation of the aortic baroreceptor reflex in the nucleus tractus solitarii of the rat, Neurosci. Lett., 89 (1988) 156-160. 14 Kubo, T., Kihara, M., Hata, H. and Misu, Y., Cardiovascular effects in rats of alpha 1 and alpha z adrenergic agents injected into the nucleus tractus solitarii, Naunyn-Schmiedeberg's Arch. Pharmacol., 335 (1987) 274-277. 15 Kubo, T. and Misu, Y., Pharmacological characterisation of the

317

16 17

18 19

20 21 22

a-adrenoceptors responsible for a decrease of blood pressure in the nucleus tractus solitarii of the rat, Naunyn-Schmiedeberg's Arch. Pharmacol., 317 (1981) 120-125. Mouchet, P., Guerin, B. and Feuerstein, C., Dissociate destruction of noradrenaline and dopamine descending projections in the thoracic spinal cord of the rat, Life Sci., 30 (1982) 373-381. Palkovits, M. and Jacobowitz, D.M., Topographic atlas of catecholamine and acetylcholinesterase-containing neurons in the rat brain. II. Hindbrain (mesencephalon, rhombencephaIon), J. Comp. Neurol., 157 (1974) 29-42. Sapru, H.N. and Krieger, A.J., Carotid and aortic chemoreceptor function in the rat, J. Appl. Physiol., 42 (1977) 344-348. Snyder, D.W., Nathan, M.A. and Reis, D.J., Chronic lability of arterial pressure produced by selective destruction of the catecholamine innervation of the nucleus tractus solitarii in the rat, Circ. Res., 43 (1978) 662-671. Starke, K. and Endo, T., Presynaptic a-adrenoceptors, Gen. Pharmacol., 7 (1976) 307-312. Takahashi, Y., Satoh, K., Sakumoto, T., Tohyama, M. and Shimizu, N., A major source of catecholamine terminals in the nucleus tractus solitarii, Brain Research, 172 (1979) 372-377. Talman, W.T., Perrone, M.H. and Reis, D.J., Evidence for L-glutamate as the neurotransmitter of baroreceptor afferent nerve fibers, Science, 209 (1980) 813-815.

23 Taiman, W.T., Snyder, D. and Reis, D.J., Chronic lability of arterial pressure produced by destruction of A2 catecholaminergic neurons in rat brainstem, Circ. Res., 46 (1980) 842-853. 24 Tung, C.-S., Onuora, C.O., Robertson, D. and Goldberg, M.R., Hypertensive effect of yohimbine following selective injection into the nucleus tractus solitarii of normotensive rats, Brain Research, 277 (1983) 193-195. 25 Ueda, H., Goshima, Y., Kubo, T. and Misu, Y., Involvement of epinephrine in the presynaptic beta adrenoceptor mechanism of norepinephrine release from rat hypothalamic slices, J. Pharmacol. Exp. Ther., 232 (1985) 507-512. 26 Unnerstall, J.M., Kopajtic, T.A. and Kuhar, M.J., Distribution of a 2 agonist binding sites in the rat and human central nervous system: analysis of some functional, anatomic correlates of the pharmacologic effects of clonidine and related adrenergic agents, Brain Res. Rev., 7 (1984) 69-101. 27 U'Prichard, D.C., Reisine, T.D., Mason, S.T., Fibiger, H.C. and Yamamura, H.I., Modulation of rat brain a- and fladrenergic receptor populations by lesion of the dorsal noradrenergic bundle, Brain Research, 187 (1980) 143-154. 28 Zandberg, D., De Jong, W. and De Wied, D., Effect of catecholamine-receptor stimulating agents on blood pressure after local application in the nucleus tractus solitarii of the medulla oblongata, Eur. J. Pharmacol., 55 (1979) 43-56.

Evidence that endogenous catecholamines are involved in alpha 2-adrenoceptor-mediated modulation of the aortic baroreceptor reflex in the nucleus tractus solitarii of the rat.

Microinjections of alpha-methylnoradrenaline and tyramine into the rat nucleus tractus solitarii (NTS) potentiated the depressor and bradycardiac resp...
430KB Sizes 0 Downloads 0 Views