Brain Research, 577 (1992) 161-164 © 1992 Elsevier Science Publishers B.V. All rights reserved. 0006-8993/92/$05.00
Angiotensin II excites vasomotor neurons but not respiratory neurons in the rostral and caudal ventrolateral medulla Yu-Wen Li, J.W. Poison and R.A.L. Dampney Department of Physiology, University of Sydney, Sydney, N.S.W. (Australia) (Accepted 7 January 1992)
Key words: Angiotensin; L-Glutamate; Ventrolateral medulla; Vasomotor activity; Respiratory activity
We examined the vasomotor and respiratory effects of angiotensin II microinjection into the rabbit ventrolateral medulla (VLM). Angiotensin II in the rostral and caudal VLM increased and decreased arterial pressure, respectively, but had no effect on phrenic nerve activity. In contrast, L-glutamate injections into the same areas altered both arterial pressure and pbrenic nerve activity. The results suggest that angiotensin II may activate specifically vasomotor neurons but not respiratory neurons in the VLM. The ventrolateral medulla (VLM) contains neurons which play a critical role in the tonic and reflex control of sympathetic vasomotor activity 7,11,w. In the rostral VLM the vasomotor neurons are sympathoexcitatory 9'~°' 22,25, while in the caudal VLM they are sympathoinhibitory 6'12'25. Both rostral and caudal parts of the VLM also contain respiratory neurons 15"2~, which are located close to or partly intermingled with vasomotor neurons TM 14. As a result, chemical stimulation of the rostral and caudal VLM by neuroexcitatory amino acids such as D,Lhomocysteate or L-glutamate affects both arterial pressure and respiratory activity, even when injected into these regions in very small volumes 5'sA2'18'19. Previous work has indicated that angiotensin II activates vasomotor neurons in the VLM 3. A high density of angiotensin II receptor binding sites has been demonstrated in those parts of the rostral and caudal VLM where vasomotor neurons are concentrated L3'2°. Microinjection of angiotensin II into the rostral VLM, or its application to the nearby ventral surface increases arterial pressure and sympathetic vasomotor nerve activity 1" 4.23. Conversely, injection of angiotensin II into the caudal V L M decreases arterial pressure and sympathetic vasomotor nerve activity 2'23. It is not known, however, whether angiotensin II in the VLM selectively activates vasomotor neurons or, like neuroexcitatory amino acids, also excites respiratory neurons. The purpose of this study was to answer this question by determining the effects on arterial pressure and phrenic nerve activity (PNA) of microinjections of angiotensin II into the rostral and caudal VLM of the rabbit. Experiments were carried out on 8 New Zealand
White rabbits (2.7-3.5 kg) anesthetized with sodium pentobarbitone (35 mg/kg i.v. followed by continuous infusion at the rate of 8-12 mg/kg/h). A tracheotomy was performed and a femoral artery and vein were catheterized. The carotid sinus, aortic and vagal nerves w e r e sectioned, in order to prevent secondary effects on arterial pressure or P N A arising from baroreceptor reflexes. A n occipital craniotomy was performed to expose the dorsal surface of the medulla oblongata. Three rabbits were paralysed with alcuronium chloride (0.1 mg/kg i.v. every 2-3 h) and artificially ventilated. In these animals the end-tidal CO 2 was monitored and maintained in the range 3-5%. Body temperature was monitored and maintained in the range 38-390°C with a thermoregulated heating lamp. Arterial pressure was recorded, and the mean arterial pressure (MAP) and heart rate measured by means of a low-pass filter and rate meter, respectively. The right phrenic nerve was exposed in the neck and P N A recorded with a paired platinum electrode. The multi-axonal discharge of the nerve was filtered, amplified and rectified. All signals were recorded on electromagnetic tape and displayed on a polygraph chart recorder. Monosodium L-glutamate (Sigma Chemical Co.) and angiotensin II (Peninsula Labs.) were dissolved in Ringer or physiological saline (pH 7.4) solution. The angiotensin II solution contained 0.2% bovine serum albumin. The concentrations of angiotensin II solution used were 1 and 20 mM, and of L-glutamate were 2 and 20 mM. Microinjections were made from either double- or single-barrelled glass pipettes into the rostral VLM pressor area and into more dorsal sites. When a single-barrelled
Correspondence: Y.W. Li, Department of Physiology, F13, University of Sydney, Sydney, N.S.W. 2006 Australia. Fax: (61) (2) 692 2058.
162 DEPTH OF PIP*'] t ~. TIP
(ram) lm ] MAP MAP
L-glutamate (20raM, ZOnl)
] s,,mm ,s,sm, ,,,,s,,m MAP
An$iolensinII (lmM, 20nl)
As1#a~la II lllOmM, ~ 1 )
Fig. 1. Effects on arterial pressure and phrenic nerve activity of microinjections of L-glutamate and angiotensin II into 3 sites, at different depths below the dorsal surface, in a track through the rostral ventrolateral medulla. Abbreviations in this and other figures: AP, arterial pressure; MAP, mean arterial pressure; PNA, phrenic nerve activity.
Fig. 3. Effects on arterial pressure and phrenic nerve activity elicited by microinjections of L-glutamate, and angiotensin II in a high concentration (20 mM) into the rostral (A) and caudal (B) ventrolateral medulla.
pipette was used, the pipette was w i t h d r a w n following L-glutamate m i c r o i n j e c t i o n a n d replaced by a n o t h e r pipette filled with a n g i o t e n s i n II solution, which was then
pressor regions of the rostral V L M increased M A P (by 36 + 6 m m H g , m e a n + S . E . M . , n = 14). In 11 of the 14 cases, the pressor responses were a c c o m p a n i e d by an
injected into sites with the same stereotaxic c o o r d i n a t e s
inhibition of respiratory activity, as o b s e r v e d either by a t e m p o r a r y a b o l i t i o n (Fig. 1) or r e d u c t i o n (Fig. 3) in peak P N A . In o n e case, P N A was increased a n d in the re-
as the g l u t a m a t e injection sites. T h e same p r o c e d u r e was used for m a k i n g m i c r o i n j e c t i o n s of L-glutamate or angiotensin II into the depressor region in the caudal V L M . Microinjections of L-glutamate (20 m M , 20 nl) into the
MAP AP MAP
PNA L-81utamate (20raM, 20hi)
~t L-glutamale (2mM, 20nl)
100 ] 5o
1oo I 50
Illllllhil,liilllllhUUlUllllUlllllllll} .llllllllll,l*lilllfllUlllllllUlllllllllllll Angiotensin II
~ Angiotensln It (ImM, 20hi)
Fig. 2. Effects on arterial pressure and phrenic nerve activity of microinjections of L-glutamate and angiotensin II into the rostral (A) and caudal (B) ventrolateral medulla.
~t A n s i o l e ~ i n n (ll~MI, ~ 1 )
Fig. 4. Effects on arterial pressure and phrenic nerve activity elicited by microinjections of l.-glutamate and a large volume (200 nl) of angiotensin II into the rostral ventrolateral medulla.
163 maining two cases it did not change. Injections of L-glutamate into more dorsal sites in the rostral V L M elicited greater effects on PNA, but the peak pressor responses were smaller and were delayed (Fig. 1). Injection of a lower concentration (2 mM, 20 nl) of L-glutamate elicited pressor responses that were much smaller (8-14 m m H g , n = 3), but these were still accompanied by a clear inhibition of P N A (Fig. 2A). The evoked changes in P N A were similar whether L-glutamate was injected into the rostral V L M on the side ipsilateral or contralateral to the site of phrenic nerve recording. Microinjections of angiotensin II (1 raM, 20 nl) into the rostral V L M also increased M A P (by 16 + 2 m m H g , n = 12), although the timecourse of the response was slower than that following L-glutamate injection, as has been previously described 23. In all cases, however, microinjection of angiotensin II had no detectable effect on P N A (Figs. 1 and 2A). Microinjection into more dorsal sites also did not cause any change in PNA, in contrast to the marked inhibition elicited by L-glutamate at the same sites (Fig. 1). Similarly, injection of angiotensin II in a concentration 20 times higher (20 mM) or volume 10 times larger (200 nl) also had no detectable effect on P N A (Figs. 3A and 4), although the pressor responses in these cases were relatively large (30-50 mmHg). Microinjection of L-glutamate (20 mM, 20 nl) into the caudal V L M decreased M A P (by 31 + 3 m m H g , n = 14), accompanied by variable effects on respiratory activity. Peak P N A was increased in 6 cases, decreased in 5 cases (Fig. 2B), and was first decreased then increased in two cases. In the remaining case, the peak activity declined but respiratory rate increased (Fig. 3B). Angiotensin II (1 mM, 20 nl) in the caudal VLM also decreased M A P (by 21 + 2 m m H g , n = 7), but in all cases again there was no detectable effect on P N A (Fig. 2B). Similarly, injection of a much higher concentration of angiotensin II (20 mM) also had no detectable effect on PNA, although it produced a large and prolonged decrease in arterial pressure (Fig. 3B). Injection of an-
giotensin II into more dorsal sites in the caudal VLM did not affect either M A P or PNA. Our results confirm previous observations in the cat and rat 5's'12'ts'19 that the pressor and depressor responses elicited by microinjection of L-glutamate into the rostral and caudal VLM, respectively, are usually accompanied by changes in respiratory activity. The main new finding of this study, however, is that microinjections of angiotensin II into the rostral and caudal V'LM had no effect on respiratory activity, although, in confirmation of previous studies 1'2'4'23, they did elicit pressor and depres-
1 Allen, A.M., Dampney, R.A.L. and Mendelsohn0 EA.O., Angiotensin receptor binding and pressor effects in cat subretrofacial nucleus, Am. J. Physiol., 255 (1988) H1011-H1017. 2 Allen, A.M., Mendelsohn, EA.O., Gieroba, Z.J. and Blessing, W.W., Vasopressin release following microinjection of angiotensin II into the caudal ventrolateral medulla oblongata in the anaesthetized rabbit, J. Neuroendocrinol., 2 (1990) 867-873. 3 Allen, A.M., Sasaki, S., Dampney, R.A.L., Mendelsohn, EA.O. and Blessing, W.W., Actions of angiotensin II in the ventrolateral medulla oblongata. In G. Kunos, J. Ciriello (Eds.).
5 Baradziej, S. and Trzebski, A., Specific areas of the ventral medulla controlling sympathetic and respiratory activities and their functional synchronization in the rat. In J. Ciriello, M.M. Caverson and C. Polosa (Eds.): The Central Neural Organiza-
Central Neural Mechanisms in Cardiovascular
Springer, New York, 1991, 95-103. 4 Andreatta, S.H., Averill, D.B., Santos, R.A.S. and Ferrario, C.M., The ventrolateral medulla: a new site of action of the renin-angiotensin system, Hypertension, 11 Suppl. i (1988) 1-1631-166.
sor responses, respectively, from these regions. No effect on P N A was observed even when the volume or concentration of angiotensin II injected was increased 10- or 20-fold. In contrast, microinjections of very small amounts (40 pmol) of L-glutamate into the VLM still elicited significant respiratory effects, although the evoked changes in arterial pressure in these cases were small. It therefore seems most unlikely that the lack of effect on P N A by angiotensin II is because respiratory neurons have a higher threshold of stimulation than vasomotor neurons in the VLM. We conclude, then, that the receptors in the VLM activated by angiotensin II are associated specifically with vasomotor neurons but not respiratory neurons in the rostral and caudal VLM. A recent study using the in vitro slice preparation has shown that putative vasomotor sympathoexcitatory neurons in the rostral VLM, which have characteristics of 'pacemaker' cells, do not respond to angiotensin II 24. It is therefore possible that the selectivity of action of angiotensin II is even greater than that revealed by the present study. For example, angiotensin II may act only on nonpacemaker sympathoexcitatory neurons in the RVLM, which include the C 1 adrenergic neurons 16. Further studies are needed, however, to answer this question.
The work was supported by the National Health and Medical Research Council of Australia.
tion of Cardiovascular Control. Progress in Brain Research, Vol. 81, Elsevier, Amsterdam, 1989, pp. 193-204. 6 Blessing, W.W. and Reis, D.J., Inhibitory cardiovascular func-
tion of neurons in the caudal ventrolateral medulla of the rabbit: relationship to the area containing A1 noradrenergic cells, Brain Res., 253 (1982) 161-171. 7 Blessing, W.W. and Li, Y.W., Inhibitory vasomotor neurons in the caudal ventrolateral region of the medulla oblongata. In J. Ciriello, M.M. Caverson and C. Polosa (Eds.), The Central Neural Organization of Cardiovascular Control. Progress in Brain Research, Vol. 81, Elsevier, Amsterdam, 1989, pp. 83-
164 8 Bonham, A.C. and Jeske I., Cardiorespiratory effects of Dt-homocysteic acid in caudal ventrolateral medulla, Am. J. Physiol., 256 (1989) H688-H696. 9 Dampney, R.A.L., Goodchild, A.K., Robertson, L.G. and Montgomery, W., Role of ventrolateral medulla in vasomotor regulation: a correlative anatomical and physiological study, Brain Res., 249 (1982) 223-235. 10 Dampney, R.A.L., Goodchild, A.K. and Tan, E., Vasopressor neurons in the rostral ventrolateral medulla of the rabbit, J. Auton. Nerv. Syst., 14 (1985) 239-254. 11 Dampney, R.A.L., The subretrofacial nucleus: its pivotal role in cardiovascular regulation, News Physiol. Sci., 5 (1990) 6367. 12 Dembowsky, K., Czachurski, J. and Seller, H., Some properties of the sympathoinhibition from the caudal ventrolateral medulla in the cat. In J. Ciriello, M.M. Caverson and C. Polosa (Eds.), The Central Neural Organization of Cardiovascular Control. Progress in Brain Research, Vol. 81, Elsevier, Amsterdam, 1989, pp. 143-157. 13 Ellenberger, H.H., Feldman, J.L. and Zhan, W.-Z, Subnuclear organization of the lateral tegmental field in the rat. II: Catecholamine neurons and ventral respiratory group, J. Comp. Neurol., 294 (1990) 212-222. 14 Ezure, K., Manabe, M. and Yamada, H., Distribution of medullary respiratory neurons in the rat, Brain Res., 455 (1988) 262-270. 15 Feldman, J.L. and Ellenberger, H.H., Central coordination of respiratory and cardiovascular control in mammals, Annu. Rev. Physiol., 50 (1988) 593-606. 16 Haselton, J.R. and Guyenet, P.G., Electrophysiological characterization of putative C~ adrenergic neurons in the rat, Neuroscience, 30 (1989) 199-214. 17 Kumada, M., Terui, N. and Kuwaki, T., Arterial baroreceptor
reflex: its central and peripheral neural mechanisms, Prog.
Neurobiol., 35 (1990) 331-361. 18 McAIlen, R.M., Location of neurones with cardiovascular and respiratory function, at the ventral surface of the cat's medulla, Neuroscience, 18 (1986) 43-49. 19 McCrimmon, D.R., Feldman, J.L. and Speck, D.E, Respiratory motoneuronal activity is altered by injections of picomoles of glutamate into cat brainstem, J. Neurosci., 6 (1986) 23842392. 20 Mendelsohn, EA.O., Allen, A.M., Clevers, J., Denton, D.A., Tarjan, E. and McKinley, M.J., Localization of angiotensin II receptor binding in rabbit brain by in vitro autoradiography, J. Cornp. Neurol., 270 (1988) 372-384. 21 Millhorn, D.E. and Eldridge, EL., Role of ventrolateral medulla in regulation of respiratory and cardiovascular systems, J. Appl. Physiol., 61 (1986) 1249-1263. 22 Ross, C.A., Ruggiero, D.A., Park, D.H., Joh, T.H., Sved, A.E, Fernandez-Pardal, J., Saavedra, J.M. and Reis, D.J., Tonic vasomotor control by the rostral ventrolateral medulla: effect of electrical or chemical stimulation of the area containing C~ adrenaline neurons on arterial pressure, heart rate and plasma catecholamines and vasopressin, J. Neurosci., 4 (1984) 479-494. 23 Sasaki, S. and Dampney, R.A.L., Tonic cardiovascular effects of angiotensin II in the ventrolateral medulla, Hypertension, 15 (1990) 274-283. 24 Sun, M.-K. and Guyenet, P.G., Effects of vasopressin and other neuropeptides on rostral medullary sympathoexcitatory neurons 'in vitro', Brain Res., 492 (1989) 261-270. 25 Willette, R.N., Barcas, P.P., Krieger, A.J. and Sapru, H.N., Vasopressor and depressor areas in the rat medulla, Neuropharmacology, 22 (1983) 1071-1079.