The Role of Central Catecholamines in the Control of Blood Pressure through the Baroreceptor Reflex and the Nasopharyngeal Reflex in the Rabbit J.P. CHALMERS, S.W. WHITE, J.B. GEFFEN and R. RUSH Departments of Medicine and Physiology, Flinders Medical Centre, Bedford Park, Adelaide, South Australia 5042 (Australia)
INTRODUCTION There is now a great deal of evidence that central catecholaminergic neurones participate in the regulation of arterial blood pressure, both in normal animals and in animals with experimental hypertension (Chalmers, 1975). In particular there is good evidence for participation of these neurones in baroreflex control of blood pressure and in the development of neurogenic hypertension (Chalmers and Wurtman, 1971; Chalmers and Reid, 1972; Doba and Reis, 1974; Chalmers, 1975). In this paper we will briefly review the role of central catecholamines in baroreceptor control of pressure and then describe more recent experiments from our laboratory on the role of central catecholaminergic nerves in the control of blood pressure through the nasopharyngeal reflex. CENTRAL CATECHOLAMINERGIC NERVES AND ARTERIAL BARORECEPTOR REFLEXES The arterial baroreceptor reflex is illustrated schematically in Fig. 1. The reflex functions as a homoeostatic mechanism regulating arterial pressure through a negative feedback loop which acts t o minimise any change in pressure and return arterial pressure back towards its set-point (Korner, 1971). This negative feedback mechanism depends upon the presence of inhibitory neurones between the afferent and efferent limbs of the reflex (Fig. 1).The afferent neurones arise from the carotid sinus and aortic arch and make their primary synapse in the nucleus of the tractus solitarii (NTS). The efferent limb effectively begins with bulbospinal neurones having their cell bodies in the brain stem in various “vasomotor” areas (VMC) and terminating in the intermediolateral cell columns of the spinal cord. Here, the bulbospinal neurones synapse either directly, or indirectly through short interneurones, with the sympathetic preganglionic neurones which pass out in the thoracolumbar outflow of the peripheral autonomic system (Fig. 1).The pathway between NTS and the vasopressor areas (or VMC) is polysynaptic, and it is within this polysynaptic pathway that the inhibitory neurones are located. For
86
-
c f=-
FACILITATORY NEURONES INHIBITORY NEURONES
Fig. 1. Simplified schematic representation of baroreflex connections showing afferent nerves from arterial baroreceptors making their primary synapse in the nucleus of the tractus solitarii (NTS), inhibitory neurones from the NTS t o the vasomotor centre (VMC), descending facilitatory bulbospinal vasomotor neurones and sympathetic preganglionic nerves. Connections from the NTS t o t h e vagal nuclei and efferent vagal fibres are also shown. Facilitatory neurones are black and inhibitory neurones are white. The half black-half white neurones connecting with the NTS and t h e VMC represent suprabulbar fibres which could be either inhibitory or facilitatory. The plus and minus signs indicate the reciprocal relationship between afferent traffic and efferent sympathetic activity. The pluses indicate an increase in activity and the minuses a decrease (Chalmers, 1975).
the sake of schematic simplicity this is drawn t o show one inhibitory neurone only in Fig. 1. There is increasing evidence for modulation of baroreceptor reflex function from connections with higher centres, both ascending and descending (Korner, 1971; Doba and Reis, 1974). Activity in afferent fibres from arterial baroreceptors, stimulated by a rise in pressure, provides a major source of inhibition of central vasomotor tone and hence of peripheral sympathetic vasomotor activity. Deafferentation of the arterial baroreceptors eliminates this inhibition and hence causes an increase in sympathetic activity and an increase in pressure, accompanied by tachycardia (Heymans and Neil, 1958; De Quattro et al., 1969; Korner, 1971). The pathways followed by central catecholaminergic nerves are similar t o those of neurones subserving central cardiovascular control (Chalmers, 1975), and it would seem reasonable t o suggest that the neurones participating in central cardiovascular control d o in fact utilise catecholamines as neurotransmitters. This concept is supported by experiments in rabbits which show that sinoaortic denervation produces a selective increase in noradrenaline
87 turnover in the hypothalamus and the thoracolumbar cord, measured by the rate of disappearance of intracisternally administered tritiated noradrenaline (Chalmers and Wurtman, 1971). The increase in hypothalamic noradrenaline turnover in this situation is consistent with the concept that baroreflex function is not mediated only at medullary level, but in fact utilises neural loops involving higher centres. The selective increase in noradrenaline turnover in the thoracolumbar cord (there was no significant change in turnover rate in cervical or lumbar segments) is consistent with an increase in the activity of bulbospinal noradrenergic nerves terminating in the lateral sympathetic horn. It should be noted that there are no monoaminergic cell bodies in the spinal cord (Dahlstrom and Fuxe, 1965) so that changes in noradrenaline metabolism in this region reflect changes in activity in nerve endings of descending bulbospinal noradrenergic tracts. The observation that sinoaortic denervation accelerates noradrenaline turnover in the thoracolumbar cord has been supported by the finding that the activity of tyrosine hydroxylase, the rate limiting enzyme in catecholamine biosynthesis, is also selectively increased in this region (Chalmers and Wurtman, 1971). On the basis of these experiments it was suggested that bulbospinal catecholaminergic nerves mediate baroreceptor reflexes, and that bulbospinal vasomotor neurones utilise catecholamines as neurotransmitters. This suggestion has been strengthened by the finding that destruction of central catecholaminergic nerves, by intracisternal administration of 6-hydroxydopamine (6-OHDA), does in fact prevent and reverse the hypertension produced by sinoaortic denervation in the rabbit (Chalmers and Reid, 1972). Doba and Reis (1973, 1974) have made similar observations in a different model of neurogenic hypertension, produced by central deafferentation of the baroreflexes using stereotactic lesions of the NTS in the rat. These workers have found that intracisternal 6-OHDA prevents the inxease in pressure seen after this mode of baroreceptor deafferentation. It has also been shown that destruction of catecholaminergic nerves in the NTS, by local stereotactic injection of 6-OHDA into this nucleus, produces an increase in blood pressure lasting about 10 days (Doba and Reis, 1974). These authors have therefore suggested that, whereas bulbospinal noradrenergic nervous activity facilitates an increase in pressure, the activity of catecholaminergic nerves synapsing in the NTS depresses arterial pressure. This suggestion is further supported by experiments in which local injection of noradrenaline into the NTS decreased arterial pressure in rats (De Jong, 1974). The simplest explanation for this phenomenon might be that there are catecholaminergic nerves terminating in the NTS (originating either from higher centres such as the hypothalamus, or from within the brain stem) synapsing with the inhibitory neurones drawn in Fig. 1. CENTRAL CATECHOLAMINERGIC NERVES AND THE NASOPHARYNGEAL REFLEX In more recent experiments, we have looked in greater depth at the mechanism whereby central administration of 6-OHDA affects arterial pressure
88
and sought t o delineate in more detail some of the peripheral autonomic mechanisms mediated by reflex activation of central catecholaminergic neurones. In these experiments, we have used a different model, namely the rabbit exposed t o vaporous stimuli, which when present in the inspired air, stimulate nasopharyngeal receptors to produce marked reflex activation of peripheral sympathetic vasoconstrictor fibres. The nasopharyngeal reflex produces a response in terrestial animals such as the rabbit, which is in many ways analogous t o that seen in the diving response in marine animals (White et al., 1974, 1975). In brief, the reflex response consists of apnoea in expiration, bradycardia, little change or a rise in blood pressure and widespread peripheral vasoconstriction (White et al., 1974, 1975). Trigeminal nerve afferents originating in the nares are responsible for the apnoea, the duration of which is influenced by arterial chemoreceptor activity; the circulatory response is again mainly evoked by trigeminal afferents with a contribution from arterial baroreceptor reflexes (White et al., 1975). The cardiovascular elements of the reflex appear to be integrated mainly at bulbospinal sites with some modulation from suprabulbar areas. In the present experiments, we used intracisternal 6-OHDA (600 pg/kg) t o examine the role of central catecholaminergic mechanisms in this reflex. In order to facilitate the analysis of the cardiovascular responses, the experiments were carried out both with and without intravenous sodium pentobarbitone (30 mglkg), which is known to interfere centrally with resting and reflex vagal activity (Crocker et al., 1967). Cigarette smoke and ammonia were used t o evoke the nasopharyngeal reflex in the rabbit. Arterial pressure and heart rate were recorded through a cannula in the ear artery of the rabbit and flow was measured in the hindlimb by chronically implanted Doppler flow probes placed around the lower abdominal aorta, immediately above the bifurcation (White et al., 1974). The effects of intracisternal 6-OHDA and intravenous sodium pentobarbitone on resting cardiovascular parameters are shown in Fig. 2. In 6 rabbits the mean heart rate was 247 t 10.0 (S.E.M.) bpm and this fell t o 199 k 21.5 (S.E.M.) bpm one week after 6-OHDA ( P d i f f < 0.001). In the same rabbits intravenous sodium pentobarbitone caused a rise in heart rate from 247 t 10.0 (S.E.M.) bpm to 297 k 15.2 (S.E.M.) bpm ( P d i f f < 0.001). When intravenous sodium pentobarbitone was given t o animals previously treated with 6-OHDA, the resting heart rate rose from 199 t 21.5 to 249 k 20.2 ( P d i f f < 0.001). The resting mean arterial pressure, hindlimb flow and hindlimb conductance were not significantly altered by either 6-OHDA i.c. or sodium pentobarbitone i.v. (Fig. 2). The restoration of the resting heart rate t o 249 bpm by the injection of sodium pentobarbitone in animals pretreated with 6-OHDA suggests that the resting bradycardia induced by 6-OHDA is due in large part t o centrally mediated reduction of resting cardiac sympathetic activity; if the activity of cardiac sympathetic nerves was normal in these animals, then central suppression of vagal activity with sodium pentobarbitone might have been expected t o elevate heart rate, towards the levels seen in normal animals given this anaesthetic, viz. about 300 bpm. These results are consistent with previous experiments which suggested that the resting bradycardia induced by 6-OHDA was in part due t o decreased cardiac sympathetic activity and also in part due
89 NASOPHARYNGEAL REFLEX ASC SAL
lZO
AP mmHg
100
60 8o
Ft
1
SOD PENT
7
6 OHDA
LJ
r
0gL
ml s-ymmHg HLC
o’81 4
CT
T
006
Fig. 2. Peak changes in mean arterial pressure (AP), heart rate (HR), hindlimb flow (HLF), and hindlimb conductance (HLC) following exposure t o smoke in 6 rabbits which were sequentially tested (1)one week after intracisternal injection of 0.9% saline containing ascorbic acid (ASC SAL), (2) while anaesthetised with sodium pentobarbitone (SOD PENT, 30 mg/kg) and ( 3 ) one week after intracisternal injection of 6-hydroxydopamine (6-OHDA, 600 pg/kg). Values are means plus t h e standard error of the mean change as determined b y analysis of variance.
to destruction of central noradrenergic tracts that normally inhibit the vagus (vagal disinhibition) (Chalmers and Reid, 1972). The results of these two studies (i.e., the present studies and those of Chalmers and Reid, 1972) clearly imply that the resting bradycardia produced by 6-OHDA has a complex mechanism originating both from destruction of noradrenergic bulbospinal fibres facilitating cardiac sympathetic activity, and from destruction of central noradrenergic neurones inhibiting cardiac vagal activity. When animals inhaled cigarette smoke through the nose there was apnoea, little change in mean arterial pressure, a marked bradycardia, and a marked reduction in hindlimb blood flow and conductance (Fig. 2; left panels). Following intravenous sodium pentobarbitone the same stimulus caused transient slowing of respiration, and the reflex bradycardia was almost abolished but there was a substantial rise in arterial pressure and a significant fall in hindlimb conductance indicating that while reflex cardiac vagal activity was almost abolished, the peripheral sympathetic vasoconstrictor mechanism was still largely intact (Fig. 2; middle panels). One week after intracisternal 6-OHDA, nasopharyngeal stimulation evoked apnoea and bradycardia. However, in contrast t o the experiments described above (middle and left panels of Fig, 2), arterial blood pressure fell and the fall in hindlimb conductance was significantly attenuated (Fig. 2; right panels). These results suggest that 6-OHDA has a selective effect on central noradrenergic neurones subserving sympathetic vasoconstrictor mechanisms. In addition
90 ASC SAL
AP mmHg
SOD PENT 6 OHDA
70
50
2'5
L
0.5
L
,030
HLC ml s-ymmHg'018 .006
LJ
Fig. 3. Peak changes in the same 6 rabbits shown in Fig. 2 following exposure t o cigarette smoke (1)one week after intracisternal injection of 0.9% saline containing ascorbic acid (left panels), and ( 2 ) during anaesthesia with sodium pentobarbitone (30 mg/kg) one week after intracisternal injection of 6-hydroxydopamine (600 pg/kg). Notation is as in Fig. 2 .
it seems likely that the vagal elements of the reflex are largely intact, since although the absolute magnitude of the bradycardia is less, this has t o be interpreted in the presence of a fall in pressure in these animals (evoking a different baroreceptor response) compared t o a maintenance of pressure in normal animals (Fig. 2). In an extension of these experiments, intravenous sodium pentobarbitone was given to animals previously treated with 6-OHDA. Following nasopharyngeal stimulation in these experiments, apnoea still occurred and there was a small rise in arterial pressure and a slight fall in hindlimb conductance (Fig. 3). In other words, the cardiovascular disturbance of nasopharyngeal stimulation can be virtually eliminated by the selective attenuation of the two major autonomic components of the response, namely, the vagal mechanism by sodium pentobarbitone and the sympathetic vasoconstrictor mechanism by 6-OHDA. At the end of the experiments the animals were sacrificed, and the brains removed and dissected over ice into 5 regions - medulla-pons, midbrain, cerebellum, hypothalamus and telencephalon - and brain noradrenaline was extracted and assayed as previously described (Chalmers and Wurtman, 1971). The noradrenaline concentration in these brain regions was reduced to about 50% of control in the 6-OHDA treated animals, as previously reported in this species (Chalmers and Reid, 1972). Dopamine-beta-hydroxylase (DBH) activity and phenylethanolamine-N-methyl-transferase(PNMT) activity were also
91 TABLE I DBH AND PNMT ACTIVITIES IN BRAIN REGIONS
Telencephalon Mid brain Medulla-pons Cerebellum Spinal cord
-
DBH(p m o les/mg/hr)
PNMT(p moles/mg/hr)
Control animals (n = 6 )
6-OHDA treated animals (n = 6 )
Control animals (n = 6 )
6-OHDA treated animals (n = 6 )
156.9 f 21.1 171.1 f 24.2 474.6 f 64.0 141.2 f 1i.i 76.0 f 9.6
70.0 f 14.6 106.7 f 23.6 210.4 f 13.7 '11.7 f 16.5 15.4 f 3.7
1.11 f 0 . 1 7 0.691 If 0.05 0.478 f 0.05 0.653 ? 0.10 1.001 k 0.11
0.95 f 0.20 0.633 0.12 0.465 f 0.08 0.665 f 0.09 0.961 f 0.12
*
Values are means f S.E.M.
assayed in these 5 brain regions using the assays described by Deguchi and Barchas (1971) and Kato e t al. (1974). The data from preliminary experiments (Table 1)revealed that the activity of DBH was reduced t o about 50% of control in all regions, whereas the activity of PNMT was virtually unchanged. This confirms the suggestion of Reid (1975), that central neurones using the putative neurotransmitter, adrenaline, are relatively resistant to the actions of 6-OHDA, compared t o noradrenergic nerves. It also suggests that the central catecholaminergic nerves contributing t o the nasopharyngeal reflex utilise noradrenaline rather than adrenaline as a neurotransmitter. CONCLUSIONS Experiments on the role of central catecholaminergic nerves in neurogenic hypertension and in baroreflex control of arterial pressure have previously demonstrated that bulbospinal catecholaminergic neurones participate in the central regulation of blood pressure and form an integral component of the baroreceptor reflex arc. The present experiments, using the nasopharyngeal reflex to study cardiovascular control, further suggest that the regulation of arterial pressure involves the participation of central catecholaminergic neurones mediating peripheral sympathetic vasoconstrictor activity. Furthermore the data on DBH and PNMT activity suggest that these central catecholaminergic nerves utilise noradrenaline as a neurotransmitter. It seems possible that noradrenergic bulbospinal nerves form an essential link in the central control of blood pressure in a number of cardiovascular reflexes. SUMMARY There is good evidence that central monoaminergic nerves participate in baroreflex control of the circulation. In particular, there is evidence that
92
bulbospinal catecholaminergic fibres terminating in the lateral horns of the spinal cord form an essential element in the baroreflex arc and mediate changes in efferent sympathetic activity, Catecholaminergic nerves also appear to play a part in a brain stem depressor mechanism involving the nucleus tractus solitarii, the site of primary synapse for afferent fibres from the arterial baroreceptors. We have recently examined the role of central catecholamines in another cardiovascular reflex - the “smoke reflex”. This is a trigeminal nerve reflex that produces a pronounced sympathetic vasoconstriction and a vagally mediated bradycardia in response to cigarette smoke stimulation of the nasopharynx of the rabbit. Following intracisternal 6-hydroxydopamine (6-OHDA), the vasoconstrictor component of the response was inactivated, but the bradycardia appeared t o be unaffected. At the end of experiments, measurements were made of regional brain noradrenaline concentration, dopamine-beta-hydroxylase (DBH) activity and phenylethanolamineN-methyl-transferase (PNMT) activity. In animals receiving 6-OHDA, noradrenaline concentration and DBH activity were reduced t o about 50% of control, but PNMT activity was unchanged. These data suggest that central pathways mediating vasoconstriction in response t o nasopharyngeal stimulation, utilise noradrenaline rather than adrenaline as a neurotransmitter. ACKNOWLEDGEMENTS This work was supported by a grant from the National Health and Medical Research Council of Australia. We would like t o thank Mrs. Soi Yen Lewis and Mrs. Lorraine Rosenberg for their expert technical assistance. REFERENCES Chalmers, J.P. (197 5 ) Brain amines and models of experimental hypertension. Circulat. Kes., 36: 469-480. Chalmers, J.P. and Reid, J.L. (1972) Participation of central noradrenergic neurones in arterial baroreceptor reflexes in t h e rabbit: a study with intracisternally administered 6-hydroxydopamine. Circulat. Res., 31 : 789-804. Chalmers, J.P. and Wurtman, R.J. (197 1)Participation of central noradrenergic neurones in arterial baroreceptor reflexes in the rabbit. Circulat. Res., 28 : 480-491. Crocker, E.F., Johnson, R.O., Korner, P.I., Uther, J.B. and White, S.W. (1968) Effects of hyperventilation o n the circulatory response of the rabbit t o arterial hypoxia. J. Physiol. (Lond.), 1 9 9 : 267-282. Dahlstrdm, A. and Fuxe, K . (1965) Evidence for t h e existence of monoamine neurones in t h e central nervous system. 11. Experimentally induced changes in the intraneuronal amine levels of bulbospinal neurone systems. Acta physiol. scand., 6 4 , Suppl. 247: 1-37. Deguchi, T. and Barchas, J. ( 1 9 7 1 ) Inhibition of transmethylation of biogenic amines by S-adenosylhomorysteine. J. biol. Chem., 246: 3175-3181. De Jong, W. ( 1 9 7 4 ) Noradrenaline: central inhibitory control of blood pressure and heart rate. Europ. J . Pharmacol., 29: 179-181. De Quattro, V., Nagatsu, T., Maronde, R. and Alexander, N. (1969) Catecholamine synthesis in rabbits with neurogenic hypertension. Circulat. Res., 24: 545-555.
93 Doba, N. and Reis, D.J. ( 1 9 7 3 ) Acute fulminating neurogenic hypertension produced b y brainstem lesions in t h e rat. Circulat. Res., 32: 584-593. Doba, N. and Reis, D.J. ( 1 9 7 4 ) Role of central and peripheral adrenergic mechanisms in neurogenic hypertension produced b y brainstem lesion in rats. Circulat. Res., 34: 293-301. Heymans, C. and Neil, E. ( 1 9 5 8 ) Reflexogenic Areas o f the Cardiovascular System, Churchill, London. Kato, T., Kuzuya, H. and Nagatsu, T. ( 1 9 7 4 ) A simple and sensitive assay for dopamine-0-hydroxylase activity by dual wavelength spectrophotometry. Biochem. Med., 1 0 : 320-328. Korner, P.I. ( 1 9 7 1 ) Integrative neural cardiovascular control. Physiol. Rev., 5 1 : 312-367. Reid, J.L. ( 1 9 7 5 ) Discussion remark. I n Central Action of Drugs in Blood Pressure Regulation, D.S. Davies and J.L. Reid (Eds.), Pitman Medical, London, p. 35. White, S.W., McRitchie, R.J. and Franklin, D.L. (1974) Autonomic cardiovascular effects of nasal inhalation of cigarette smoke in the rabbit. Aust. J. exp. Biol. med. Sci., 52: 111-126. White, S.W., McRitchie, R.J. and Korner, P.I. (1975) Central nervous system control of cardiorespiratory nasopharyngeal reflexes in the rabbit. Amer. J. Physiol., 228: 404-409.
INTRODUCTION There is now a great deal of evidence that central catecholaminergic neurones participate in the regulation of arterial blood pressure, both in normal animals and in animals with experimental hypertension (Chalmers, 1975). In particular there is good evidence for participation of these neurones in baroreflex control of blood pressure and in the development of neurogenic hypertension (Chalmers and Wurtman, 1971; Chalmers and Reid, 1972; Doba and Reis, 1974; Chalmers, 1975). In this paper we will briefly review the role of central catecholamines in baroreceptor control of pressure and then describe more recent experiments from our laboratory on the role of central catecholaminergic nerves in the control of blood pressure through the nasopharyngeal reflex. CENTRAL CATECHOLAMINERGIC NERVES AND ARTERIAL BARORECEPTOR REFLEXES The arterial baroreceptor reflex is illustrated schematically in Fig. 1. The reflex functions as a homoeostatic mechanism regulating arterial pressure through a negative feedback loop which acts t o minimise any change in pressure and return arterial pressure back towards its set-point (Korner, 1971). This negative feedback mechanism depends upon the presence of inhibitory neurones between the afferent and efferent limbs of the reflex (Fig. 1).The afferent neurones arise from the carotid sinus and aortic arch and make their primary synapse in the nucleus of the tractus solitarii (NTS). The efferent limb effectively begins with bulbospinal neurones having their cell bodies in the brain stem in various “vasomotor” areas (VMC) and terminating in the intermediolateral cell columns of the spinal cord. Here, the bulbospinal neurones synapse either directly, or indirectly through short interneurones, with the sympathetic preganglionic neurones which pass out in the thoracolumbar outflow of the peripheral autonomic system (Fig. 1).The pathway between NTS and the vasopressor areas (or VMC) is polysynaptic, and it is within this polysynaptic pathway that the inhibitory neurones are located. For
86
-
c f=-
FACILITATORY NEURONES INHIBITORY NEURONES
Fig. 1. Simplified schematic representation of baroreflex connections showing afferent nerves from arterial baroreceptors making their primary synapse in the nucleus of the tractus solitarii (NTS), inhibitory neurones from the NTS t o the vasomotor centre (VMC), descending facilitatory bulbospinal vasomotor neurones and sympathetic preganglionic nerves. Connections from the NTS t o t h e vagal nuclei and efferent vagal fibres are also shown. Facilitatory neurones are black and inhibitory neurones are white. The half black-half white neurones connecting with the NTS and t h e VMC represent suprabulbar fibres which could be either inhibitory or facilitatory. The plus and minus signs indicate the reciprocal relationship between afferent traffic and efferent sympathetic activity. The pluses indicate an increase in activity and the minuses a decrease (Chalmers, 1975).
the sake of schematic simplicity this is drawn t o show one inhibitory neurone only in Fig. 1. There is increasing evidence for modulation of baroreceptor reflex function from connections with higher centres, both ascending and descending (Korner, 1971; Doba and Reis, 1974). Activity in afferent fibres from arterial baroreceptors, stimulated by a rise in pressure, provides a major source of inhibition of central vasomotor tone and hence of peripheral sympathetic vasomotor activity. Deafferentation of the arterial baroreceptors eliminates this inhibition and hence causes an increase in sympathetic activity and an increase in pressure, accompanied by tachycardia (Heymans and Neil, 1958; De Quattro et al., 1969; Korner, 1971). The pathways followed by central catecholaminergic nerves are similar t o those of neurones subserving central cardiovascular control (Chalmers, 1975), and it would seem reasonable t o suggest that the neurones participating in central cardiovascular control d o in fact utilise catecholamines as neurotransmitters. This concept is supported by experiments in rabbits which show that sinoaortic denervation produces a selective increase in noradrenaline
87 turnover in the hypothalamus and the thoracolumbar cord, measured by the rate of disappearance of intracisternally administered tritiated noradrenaline (Chalmers and Wurtman, 1971). The increase in hypothalamic noradrenaline turnover in this situation is consistent with the concept that baroreflex function is not mediated only at medullary level, but in fact utilises neural loops involving higher centres. The selective increase in noradrenaline turnover in the thoracolumbar cord (there was no significant change in turnover rate in cervical or lumbar segments) is consistent with an increase in the activity of bulbospinal noradrenergic nerves terminating in the lateral sympathetic horn. It should be noted that there are no monoaminergic cell bodies in the spinal cord (Dahlstrom and Fuxe, 1965) so that changes in noradrenaline metabolism in this region reflect changes in activity in nerve endings of descending bulbospinal noradrenergic tracts. The observation that sinoaortic denervation accelerates noradrenaline turnover in the thoracolumbar cord has been supported by the finding that the activity of tyrosine hydroxylase, the rate limiting enzyme in catecholamine biosynthesis, is also selectively increased in this region (Chalmers and Wurtman, 1971). On the basis of these experiments it was suggested that bulbospinal catecholaminergic nerves mediate baroreceptor reflexes, and that bulbospinal vasomotor neurones utilise catecholamines as neurotransmitters. This suggestion has been strengthened by the finding that destruction of central catecholaminergic nerves, by intracisternal administration of 6-hydroxydopamine (6-OHDA), does in fact prevent and reverse the hypertension produced by sinoaortic denervation in the rabbit (Chalmers and Reid, 1972). Doba and Reis (1973, 1974) have made similar observations in a different model of neurogenic hypertension, produced by central deafferentation of the baroreflexes using stereotactic lesions of the NTS in the rat. These workers have found that intracisternal 6-OHDA prevents the inxease in pressure seen after this mode of baroreceptor deafferentation. It has also been shown that destruction of catecholaminergic nerves in the NTS, by local stereotactic injection of 6-OHDA into this nucleus, produces an increase in blood pressure lasting about 10 days (Doba and Reis, 1974). These authors have therefore suggested that, whereas bulbospinal noradrenergic nervous activity facilitates an increase in pressure, the activity of catecholaminergic nerves synapsing in the NTS depresses arterial pressure. This suggestion is further supported by experiments in which local injection of noradrenaline into the NTS decreased arterial pressure in rats (De Jong, 1974). The simplest explanation for this phenomenon might be that there are catecholaminergic nerves terminating in the NTS (originating either from higher centres such as the hypothalamus, or from within the brain stem) synapsing with the inhibitory neurones drawn in Fig. 1. CENTRAL CATECHOLAMINERGIC NERVES AND THE NASOPHARYNGEAL REFLEX In more recent experiments, we have looked in greater depth at the mechanism whereby central administration of 6-OHDA affects arterial pressure
88
and sought t o delineate in more detail some of the peripheral autonomic mechanisms mediated by reflex activation of central catecholaminergic neurones. In these experiments, we have used a different model, namely the rabbit exposed t o vaporous stimuli, which when present in the inspired air, stimulate nasopharyngeal receptors to produce marked reflex activation of peripheral sympathetic vasoconstrictor fibres. The nasopharyngeal reflex produces a response in terrestial animals such as the rabbit, which is in many ways analogous t o that seen in the diving response in marine animals (White et al., 1974, 1975). In brief, the reflex response consists of apnoea in expiration, bradycardia, little change or a rise in blood pressure and widespread peripheral vasoconstriction (White et al., 1974, 1975). Trigeminal nerve afferents originating in the nares are responsible for the apnoea, the duration of which is influenced by arterial chemoreceptor activity; the circulatory response is again mainly evoked by trigeminal afferents with a contribution from arterial baroreceptor reflexes (White et al., 1975). The cardiovascular elements of the reflex appear to be integrated mainly at bulbospinal sites with some modulation from suprabulbar areas. In the present experiments, we used intracisternal 6-OHDA (600 pg/kg) t o examine the role of central catecholaminergic mechanisms in this reflex. In order to facilitate the analysis of the cardiovascular responses, the experiments were carried out both with and without intravenous sodium pentobarbitone (30 mglkg), which is known to interfere centrally with resting and reflex vagal activity (Crocker et al., 1967). Cigarette smoke and ammonia were used t o evoke the nasopharyngeal reflex in the rabbit. Arterial pressure and heart rate were recorded through a cannula in the ear artery of the rabbit and flow was measured in the hindlimb by chronically implanted Doppler flow probes placed around the lower abdominal aorta, immediately above the bifurcation (White et al., 1974). The effects of intracisternal 6-OHDA and intravenous sodium pentobarbitone on resting cardiovascular parameters are shown in Fig. 2. In 6 rabbits the mean heart rate was 247 t 10.0 (S.E.M.) bpm and this fell t o 199 k 21.5 (S.E.M.) bpm one week after 6-OHDA ( P d i f f < 0.001). In the same rabbits intravenous sodium pentobarbitone caused a rise in heart rate from 247 t 10.0 (S.E.M.) bpm to 297 k 15.2 (S.E.M.) bpm ( P d i f f < 0.001). When intravenous sodium pentobarbitone was given t o animals previously treated with 6-OHDA, the resting heart rate rose from 199 t 21.5 to 249 k 20.2 ( P d i f f < 0.001). The resting mean arterial pressure, hindlimb flow and hindlimb conductance were not significantly altered by either 6-OHDA i.c. or sodium pentobarbitone i.v. (Fig. 2). The restoration of the resting heart rate t o 249 bpm by the injection of sodium pentobarbitone in animals pretreated with 6-OHDA suggests that the resting bradycardia induced by 6-OHDA is due in large part t o centrally mediated reduction of resting cardiac sympathetic activity; if the activity of cardiac sympathetic nerves was normal in these animals, then central suppression of vagal activity with sodium pentobarbitone might have been expected t o elevate heart rate, towards the levels seen in normal animals given this anaesthetic, viz. about 300 bpm. These results are consistent with previous experiments which suggested that the resting bradycardia induced by 6-OHDA was in part due t o decreased cardiac sympathetic activity and also in part due
89 NASOPHARYNGEAL REFLEX ASC SAL
lZO
AP mmHg
100
60 8o
Ft
1
SOD PENT
7
6 OHDA
LJ
r
0gL
ml s-ymmHg HLC
o’81 4
CT
T
006
Fig. 2. Peak changes in mean arterial pressure (AP), heart rate (HR), hindlimb flow (HLF), and hindlimb conductance (HLC) following exposure t o smoke in 6 rabbits which were sequentially tested (1)one week after intracisternal injection of 0.9% saline containing ascorbic acid (ASC SAL), (2) while anaesthetised with sodium pentobarbitone (SOD PENT, 30 mg/kg) and ( 3 ) one week after intracisternal injection of 6-hydroxydopamine (6-OHDA, 600 pg/kg). Values are means plus t h e standard error of the mean change as determined b y analysis of variance.
to destruction of central noradrenergic tracts that normally inhibit the vagus (vagal disinhibition) (Chalmers and Reid, 1972). The results of these two studies (i.e., the present studies and those of Chalmers and Reid, 1972) clearly imply that the resting bradycardia produced by 6-OHDA has a complex mechanism originating both from destruction of noradrenergic bulbospinal fibres facilitating cardiac sympathetic activity, and from destruction of central noradrenergic neurones inhibiting cardiac vagal activity. When animals inhaled cigarette smoke through the nose there was apnoea, little change in mean arterial pressure, a marked bradycardia, and a marked reduction in hindlimb blood flow and conductance (Fig. 2; left panels). Following intravenous sodium pentobarbitone the same stimulus caused transient slowing of respiration, and the reflex bradycardia was almost abolished but there was a substantial rise in arterial pressure and a significant fall in hindlimb conductance indicating that while reflex cardiac vagal activity was almost abolished, the peripheral sympathetic vasoconstrictor mechanism was still largely intact (Fig. 2; middle panels). One week after intracisternal 6-OHDA, nasopharyngeal stimulation evoked apnoea and bradycardia. However, in contrast t o the experiments described above (middle and left panels of Fig, 2), arterial blood pressure fell and the fall in hindlimb conductance was significantly attenuated (Fig. 2; right panels). These results suggest that 6-OHDA has a selective effect on central noradrenergic neurones subserving sympathetic vasoconstrictor mechanisms. In addition
90 ASC SAL
AP mmHg
SOD PENT 6 OHDA
70
50
2'5
L
0.5
L
,030
HLC ml s-ymmHg'018 .006
LJ
Fig. 3. Peak changes in the same 6 rabbits shown in Fig. 2 following exposure t o cigarette smoke (1)one week after intracisternal injection of 0.9% saline containing ascorbic acid (left panels), and ( 2 ) during anaesthesia with sodium pentobarbitone (30 mg/kg) one week after intracisternal injection of 6-hydroxydopamine (600 pg/kg). Notation is as in Fig. 2 .
it seems likely that the vagal elements of the reflex are largely intact, since although the absolute magnitude of the bradycardia is less, this has t o be interpreted in the presence of a fall in pressure in these animals (evoking a different baroreceptor response) compared t o a maintenance of pressure in normal animals (Fig. 2). In an extension of these experiments, intravenous sodium pentobarbitone was given to animals previously treated with 6-OHDA. Following nasopharyngeal stimulation in these experiments, apnoea still occurred and there was a small rise in arterial pressure and a slight fall in hindlimb conductance (Fig. 3). In other words, the cardiovascular disturbance of nasopharyngeal stimulation can be virtually eliminated by the selective attenuation of the two major autonomic components of the response, namely, the vagal mechanism by sodium pentobarbitone and the sympathetic vasoconstrictor mechanism by 6-OHDA. At the end of the experiments the animals were sacrificed, and the brains removed and dissected over ice into 5 regions - medulla-pons, midbrain, cerebellum, hypothalamus and telencephalon - and brain noradrenaline was extracted and assayed as previously described (Chalmers and Wurtman, 1971). The noradrenaline concentration in these brain regions was reduced to about 50% of control in the 6-OHDA treated animals, as previously reported in this species (Chalmers and Reid, 1972). Dopamine-beta-hydroxylase (DBH) activity and phenylethanolamine-N-methyl-transferase(PNMT) activity were also
91 TABLE I DBH AND PNMT ACTIVITIES IN BRAIN REGIONS
Telencephalon Mid brain Medulla-pons Cerebellum Spinal cord
-
DBH(p m o les/mg/hr)
PNMT(p moles/mg/hr)
Control animals (n = 6 )
6-OHDA treated animals (n = 6 )
Control animals (n = 6 )
6-OHDA treated animals (n = 6 )
156.9 f 21.1 171.1 f 24.2 474.6 f 64.0 141.2 f 1i.i 76.0 f 9.6
70.0 f 14.6 106.7 f 23.6 210.4 f 13.7 '11.7 f 16.5 15.4 f 3.7
1.11 f 0 . 1 7 0.691 If 0.05 0.478 f 0.05 0.653 ? 0.10 1.001 k 0.11
0.95 f 0.20 0.633 0.12 0.465 f 0.08 0.665 f 0.09 0.961 f 0.12
*
Values are means f S.E.M.
assayed in these 5 brain regions using the assays described by Deguchi and Barchas (1971) and Kato e t al. (1974). The data from preliminary experiments (Table 1)revealed that the activity of DBH was reduced t o about 50% of control in all regions, whereas the activity of PNMT was virtually unchanged. This confirms the suggestion of Reid (1975), that central neurones using the putative neurotransmitter, adrenaline, are relatively resistant to the actions of 6-OHDA, compared t o noradrenergic nerves. It also suggests that the central catecholaminergic nerves contributing t o the nasopharyngeal reflex utilise noradrenaline rather than adrenaline as a neurotransmitter. CONCLUSIONS Experiments on the role of central catecholaminergic nerves in neurogenic hypertension and in baroreflex control of arterial pressure have previously demonstrated that bulbospinal catecholaminergic neurones participate in the central regulation of blood pressure and form an integral component of the baroreceptor reflex arc. The present experiments, using the nasopharyngeal reflex to study cardiovascular control, further suggest that the regulation of arterial pressure involves the participation of central catecholaminergic neurones mediating peripheral sympathetic vasoconstrictor activity. Furthermore the data on DBH and PNMT activity suggest that these central catecholaminergic nerves utilise noradrenaline as a neurotransmitter. It seems possible that noradrenergic bulbospinal nerves form an essential link in the central control of blood pressure in a number of cardiovascular reflexes. SUMMARY There is good evidence that central monoaminergic nerves participate in baroreflex control of the circulation. In particular, there is evidence that
92
bulbospinal catecholaminergic fibres terminating in the lateral horns of the spinal cord form an essential element in the baroreflex arc and mediate changes in efferent sympathetic activity, Catecholaminergic nerves also appear to play a part in a brain stem depressor mechanism involving the nucleus tractus solitarii, the site of primary synapse for afferent fibres from the arterial baroreceptors. We have recently examined the role of central catecholamines in another cardiovascular reflex - the “smoke reflex”. This is a trigeminal nerve reflex that produces a pronounced sympathetic vasoconstriction and a vagally mediated bradycardia in response to cigarette smoke stimulation of the nasopharynx of the rabbit. Following intracisternal 6-hydroxydopamine (6-OHDA), the vasoconstrictor component of the response was inactivated, but the bradycardia appeared t o be unaffected. At the end of experiments, measurements were made of regional brain noradrenaline concentration, dopamine-beta-hydroxylase (DBH) activity and phenylethanolamineN-methyl-transferase (PNMT) activity. In animals receiving 6-OHDA, noradrenaline concentration and DBH activity were reduced t o about 50% of control, but PNMT activity was unchanged. These data suggest that central pathways mediating vasoconstriction in response t o nasopharyngeal stimulation, utilise noradrenaline rather than adrenaline as a neurotransmitter. ACKNOWLEDGEMENTS This work was supported by a grant from the National Health and Medical Research Council of Australia. We would like t o thank Mrs. Soi Yen Lewis and Mrs. Lorraine Rosenberg for their expert technical assistance. REFERENCES Chalmers, J.P. (197 5 ) Brain amines and models of experimental hypertension. Circulat. Kes., 36: 469-480. Chalmers, J.P. and Reid, J.L. (1972) Participation of central noradrenergic neurones in arterial baroreceptor reflexes in t h e rabbit: a study with intracisternally administered 6-hydroxydopamine. Circulat. Res., 31 : 789-804. Chalmers, J.P. and Wurtman, R.J. (197 1)Participation of central noradrenergic neurones in arterial baroreceptor reflexes in the rabbit. Circulat. Res., 28 : 480-491. Crocker, E.F., Johnson, R.O., Korner, P.I., Uther, J.B. and White, S.W. (1968) Effects of hyperventilation o n the circulatory response of the rabbit t o arterial hypoxia. J. Physiol. (Lond.), 1 9 9 : 267-282. Dahlstrdm, A. and Fuxe, K . (1965) Evidence for t h e existence of monoamine neurones in t h e central nervous system. 11. Experimentally induced changes in the intraneuronal amine levels of bulbospinal neurone systems. Acta physiol. scand., 6 4 , Suppl. 247: 1-37. Deguchi, T. and Barchas, J. ( 1 9 7 1 ) Inhibition of transmethylation of biogenic amines by S-adenosylhomorysteine. J. biol. Chem., 246: 3175-3181. De Jong, W. ( 1 9 7 4 ) Noradrenaline: central inhibitory control of blood pressure and heart rate. Europ. J . Pharmacol., 29: 179-181. De Quattro, V., Nagatsu, T., Maronde, R. and Alexander, N. (1969) Catecholamine synthesis in rabbits with neurogenic hypertension. Circulat. Res., 24: 545-555.
93 Doba, N. and Reis, D.J. ( 1 9 7 3 ) Acute fulminating neurogenic hypertension produced b y brainstem lesions in t h e rat. Circulat. Res., 32: 584-593. Doba, N. and Reis, D.J. ( 1 9 7 4 ) Role of central and peripheral adrenergic mechanisms in neurogenic hypertension produced b y brainstem lesion in rats. Circulat. Res., 34: 293-301. Heymans, C. and Neil, E. ( 1 9 5 8 ) Reflexogenic Areas o f the Cardiovascular System, Churchill, London. Kato, T., Kuzuya, H. and Nagatsu, T. ( 1 9 7 4 ) A simple and sensitive assay for dopamine-0-hydroxylase activity by dual wavelength spectrophotometry. Biochem. Med., 1 0 : 320-328. Korner, P.I. ( 1 9 7 1 ) Integrative neural cardiovascular control. Physiol. Rev., 5 1 : 312-367. Reid, J.L. ( 1 9 7 5 ) Discussion remark. I n Central Action of Drugs in Blood Pressure Regulation, D.S. Davies and J.L. Reid (Eds.), Pitman Medical, London, p. 35. White, S.W., McRitchie, R.J. and Franklin, D.L. (1974) Autonomic cardiovascular effects of nasal inhalation of cigarette smoke in the rabbit. Aust. J. exp. Biol. med. Sci., 52: 111-126. White, S.W., McRitchie, R.J. and Korner, P.I. (1975) Central nervous system control of cardiorespiratory nasopharyngeal reflexes in the rabbit. Amer. J. Physiol., 228: 404-409.
