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Cardiovascular response to injections of enkephalin in the pressor area of the ventrolateral medulla
~~ur~~~~~~~~~~~~},Vol. 23, No. 8,
pp. 939-946.
1984
Printed in Great Britain. All rights reserved
CARDIOVASCULAR RESPONSE TO INJECTIONS ENKEPHALIN IN THE PRESSBR AREA OF THE VENTROLATERAL MEDULLA*
OF
SUSAN PUNNEN, R. WILLETTE, A. J. KRIECER and H. N. SAPRUT Department of Pharmacology and Section of Neurosurgery, University of Medicine and Dentistry of New Jersey. New Jersey Medical School, Newark, NJ 07103 and V.A. Medical Center, East Orange, NJ 07109, U.S.A.
Summary-The
cardiovascular effects of the injection of an e~kephalin analogue, [D-ala’-mets]enkephalinamide (DAME) into the pressor area of the rostra1 ventrolateral medulla were studied in urethane-anesthetized and decerebrate rats. The excitatory amino acid L-glutamate was used to identify the ventrolateral medulla. The pressor responses to t-glutamate were elicited from an area that included the nucleus reticularis gigantocellularis, the medial aspect of the nucleus reticularis parvocellularis and the dorsal portion of the nucleus reticularis lateralis. Injection (0.1 ~1 volume) of DAME (2.5-500.0 ng/site) into the ventrolateral medulla elicited a dose-related decrease in arterial blood pressure and heart rate and attenuated the carotid occlusion response (COR), Control injections (0.1-0.2~1 vol) of saline into the same area failed to produce any response. The specificity of this opiate response was tested with naloxone HCI, an opiate antagonist, which prevented, as well as reversed, the action of DAME both by intravenous (i.v.) administration and by injection into the ventro~atera~ medulla. It was concluded that the ventrolateral rn~~~~a plays a role in the generation of vasomotor tone and that stimulation of opiate receptors in this area by an e~kepha~in analogue produced hypote~sion, bradycar~~a and modi~~at~on of cardiovascular reflexes. Key words: bradycardia, carotid occlusion, enkephalin, vasotiressor neurons; ventrolateral medulla.
~ypot~nsio~, naloxone, opiate receptors.
and Rascher, 1981; Bolme, Fuxe, Agnati, Bradley and Smythies, 1978; Lang, Unger, Rascher and Ganten, 1983; Palkovits, 198 1). Among them, opioids have been shown to exert potent effects on blood pressure and they are receiving increasing attention since they have been found to be present in the cardiovascular centers of the brain (Sima~t~v, Kuhar, Uhl and Snyder, 1977; Uhl, Goodman, Kuhar, er, 1979). The effect of opiates on cardiovascular regulation in the ventrolateral vasodepressor area, i.e. the A,, area (Dahlstrom and Fuxe, 1964), was studied by Willette, Punnen, Krieger and Sapru (1984). The present study was extended to explore the significance of opiate receptor stimulation in the ventrolateral vasopressor neuron pool, which is located rostra1 to the vasodepressor area and seems to coincide with rostra! C,, epinephrine-containing neurons (H&kfelt, Fuxe, Goldstein and Johansso~, 1973). ~~-a~a~-met5~~~kepha~~~arn~de (~AM~)~ a stable analogue of enkep~alin (Pert, Pert, Cheng and Fang, 1976) was chosen for the study of the effects of opiates on blood pressure, heart rate and cardiovascular reflexes in the rostra1 ventrolateral vasopressor area.
It has become increasingly clear that the functional maintenance of cardiovascular homeostasis is dependent upon the integrity of several nuclei which are located in the medulla (Calaresu, Faiers and Mogenson, 1975). More recently the ventral medulla has been shown to play an important role in the central regulation of blood pressure (Feldberg, 1974; ~arnp~~y~ 1981; Wen~~rgr~n and Oberg, 1980). The presence of depressor and pressor areas in the medulla, regulating sympathetic activity, was demonstrated by Alexander (1946) employing electrical stimulation techniques. Since electrical stimulation evokes responses from cell bodies, fibers of passage the excitatory amino acid and nerve endings, L-glutamate was used to map out the depressor and pressor neuron pools in the ventrolateral medulla, by Krieger and Sapru (1983). Willette, Barcas, L-Glutamate is known to stimulate cell bodies and not axons of passage (Krinjevic and Phillips, 1963). Several peptides from brain have been imprecated in the central regulation of blood pressure (Ganten, Unger, Simon, Schaz, Schiilkens, Mann, Speck, Lang
*A preliminary report of this study has appeared as an abstract in Fedn hoc. Fedn Am. Sum exp. Biol. 42: 653,
METHODS
SuvgicaEprocedures
1983.
tAddress all correspondence to: Hreday N. Sapru, Ph.D., Neurosurgery, Medical Sciences Bldg, H-592, New Jersey Medical School. Newark, NJ 07103, U.S.A. 939
All experiments were carried out acutely in male Wistar rats (Royal Hart, New York) weighing be-
940
SUSAN PUNNEN at d.
tween 250 and 350 g. In the anesthetized preparation urethane (I .O- 1.2 g/kg, i.p.) was used. Experiments were also done using unanesthetized decerebrate animals, as described in detail elsewhere (Sapru and Krieger, 197X). In all experiments the trachea, as well as the femoral vein and artery. was cannulated. The animals were then placed in a stereotaxic instrument (David Kopf Instruments) in a supine position with the upper incisor bar at the level of the interaural line. The upper trachea, larynx. esophagus and surrounding musculature were excised and the longus capitis muscles were removed bilaterally, exposing the occipital foramen and the basal portion of the occipital bone. The basal portion of the occipital bone was then carefully removed. creating a ventral window approx. 6 mm wide and 7 mm long (Willette et al., 1983a). The rostrocaudal, mediolateral and dorsoventral stereotaxic coordinates were in reference to the most caudal aspect of the occipital foramen. basilar artery and ventral surface of the respectively. Upon histological exammedulla, ination. the rostrocaudal landmark was found to be 2.0-2.5 mm caudal to the obex. In all experiments, blood pressure (BP), mean arterial pressure (MAP) and heart rate (HR) were monitored. Blood pressure was recorded from the left femoral artery, using a pressure transducer (Statham P23 Db) and heart rate was monitored by a tachograph (Grass Model 7P4). The rectal temperature was monitored by a digital thermometer (BAT-X, Bailey Instruments) via a rectal probe and maintained at 37 k 0.5 C. Animals were artificially ventilated with room air, using a rodent respirator (Harvard, 680). to avoid secondary cardiovascular effects. In the animals in which the responses to carotid occlusion were elicited. the common carotid arteries were freed from the vagus and sympathetic nerves and the surrounding connective tissue. Carotid occlusion was performed with hemoclips for I5 set intervals before and after the administration of saline and DAME.
L-Glutamate monosodium (L-glu), [o-ala’-met5]enkephalinamide and other drugs used in this study were dissolved in 0.9”,, NaCl (pH 7.0) and injected in a volume of 0.1 PI over a 5 set interval. The solutions of drug were delivered through glass multibarrel micropipettes (Medical Systems, Inc., New York) pulled to an outside tip diameter of approx. 501tm. Unused barrels of the micropipette were filled with 0.9”,,, NaCl and sealed. The vasopressor areas were consistently identified in the ventrolateral medulla by injecting the excitatory amino acid L-glutamate (333 ng:‘site) into an area extending from I.5 to 2.2 mm rostra1 to the obex. I .4 to I .X mm lateral to the basilar artery and 0.X to I .3 mm from the ventral surface. At the end of each experiment, the stereotaxic coordinates of the sites of injection were noted and an
electrolytic lesion was then made by passing a constant current of 3.5 mA for 30 set through a platinum-iridium microelectrode (20 pm O.D.). The brain was then fixed by intravascular perfusion with IO”, formaldehyde. Thin coronal sections ( 12pm) ol the appropriate areas were cut, mounted on microscope slides and stained with cresyl violet for histological identification of the sites of injection. Stutislical
anal~xis
Each animal served as its own control and a paired two-tailed r-test was used to determine whether the mean from a population of differences was zero (Croxton, 1959). Independent f-test was used to test the significance of differences. if any, between the cardiovascular responses, in decerebrate and in urethane-anesthetized rats. Differences were considered significant at P < 0.05. All values are expressed as the mean k the standard error of the mean (SEM). RESULTS
The vasopressor sites in the ventrolateral medulla were identified (Fig. IA) bilaterally by injecting L-glutamate (333 ngjsite). L-Glutamate produced an increase in blood pressure and heart rate. When DAME (50 ng/site) was injected into these sites, the most striking effect was a decrease in blood pressure. Decreases in pulse pressure and heart rate were also observed. The onset of action of this response was immediate (Fig. IB). The histological sites from which DAME elicited the depressor response are shown in Fig. IC. Earlier studies have shown that the vasopressor sites were located in the lateral border of the nucleus reticularis gigantocellularis, the medial aspect of the nucleus reticularis parvocellularis and the dorsal portion of the nucleus reticularis lateralis (Willette et cd., 1983a). After identification of the vasopressor sites with L-glutamate several doses of DAME were injected into these sites to study dose--response relationships. Since tachyphylaxis to repeated administration of DAME was evident. different doses were administered at intervals of 45 min. A dose range of 2.5 to 500.0 ngjsite of DAME was used. The duration and magnitude of the responses in blood pressure and heart rate to DAME were found to be dose-related. A total of 5 animals was used in this study. The control blood pressure and heart rate in the animals was 92 k 4 mmHg and 3X7 + I4 bpm. respectively. Figure 2A shows the semilogarithmic dose-response curve for changes in blood pressure with injections 01 DAME. The fall in blood pressure was I5 + 2 mmHg following 2.5 ng:‘site and, 49 + 3 mmHg a1 lOOng/site. The peak etfect on the blood pressure response was seen at the dose of 100 ng/site; increasing the dose to 500 ng/site did not elicit a furthei decrease in blood pressure. The change in heart rate
Opiate action in medullary pressor area PRESSOR (A)
941
AREA
right
left
OL 600
g 200
[
200
ik
LLW 0
! t L-Oh
+ L-Ok
1333 ngf
(B)
u33
ng)
(Cl
200
0 600
[ r
OL t DAME
right(SOng)
t left (Song)
I
#
1 MIN
Fig. 1. In each panel, tracings from top to bottom are of mean arterial pressure (MAP). heart rate (HR), and pulse pressure (BP). The volume of injection of L-glutamate (L-glu, 333 ng) and DAME (50 ng) was 0. I pl. Panel A: typical L-glutamate induced rise in blood pressure and heart rate, after injection into right and left side of the ventrolateral medulla. Panel B: decrease in blood pressure and heart rate following the bilateral injection of DAME into the same sites indicated by the arrows. Control injections of 0. I to 0.2~11of saline produced no responses. Panel C: filled circles indicate the medullary site which elicited the responses of L-glutamate and DAME. Abbreviations: na, nucleus ambiguus; ngc, nucleus gigantocellularis; nrl, nucleus reticularis lateralis; nts, nucleus trdctus solitarius; ntv, nucleus tractus spinalis nervi trigemini; p. pyramidal tract; rpc, nucleus reticularis parvocehukdris.
with DAME was less dramatic than the blood pressure response. As shown in Fig. 2B, at 2.5 ng/site a decrease of 20 + 4 bpm was observed and a maximal decrease of 40 + 5 beats was seen at 100 ng/site of DAME. The duration of action of DAME on blood pressure ranged from 6 to 8 min at 50ng/site to a more prolonged action of I5 to 20 min at 500 ngjsite. To show that changes in blood pressure and heart rate associated with the injection of DAME into the vasopressor area were opiate specific, naloxone, an was administered both intraopiate antagonist, venously (I mg/kg, iv.) and by injection (2 pg/site) into the vasopressor sites. A total of 7 animals was used in this study. The control blood pressure and
heart rate in the animals was 92 + 2 mmHg and 417 + 19 bpm, respectively. After injection of DAME (50ng/site) the fall in blood pressure was 46 f 4mmHg and the heart rate decreased 34 + 3 bpm. Reversal of the cardiovascular effects of DAME to control levels was observed at both routes of administration (i.v. and injection) of naloxone which, by itself, has no effect on blood pressure and heart rate. Figure 3A shows the effect of DAME on blood pressure and heart rate and its reversal by naloxone. Control mean arterial blood pressure (MAP) was 85mmHg and decreased to 35mmHg after bilateral administration of DAME (50 ngjsite), a decrease of 50mmHg; the heart rate (control
942
SUSAN PUNNEN
(A)
.i-
I
25
I
1
50
I
1
I
250 500 1000 DAME
5000
1
(ng/slte
(6)
E a 0
0 -10
-I -20 "I L: -30
i\: ---d
g -40 6 Jz 0
1
25
I
50
I
T I---.----_ 1 I
I
I
25.0 500 1000
DAME (ng /site
5000
1
Fig. 2. Dose-response curves for depressor and bradycardic responses. Panel A: depressor responses after the bilateral injection of DAME into the ventrolateral vasopressor area. Doses ranging from 2.5 to 500.0 ng were used. Maximal responses were seen with the 100 ng/site. Panel B: decrease in heart rate (HR) following injection of DAME into the ventrolateral medulla at the various doses. All values are expressed as mean + SEM (n = 5).
430 bpm) decreased by 20 bpm. Naloxone (1 mg/kg. i.v.) given 7 min after the administration of DAME immediately reversed blood pressure to control levels. Injection of naloxone into the same vasopressor sites at the peak effect of DAME on blood pressure (60 mmHg decrease) reversed the blood pressure response (Fig. 3B). The time course of the reversal of blood pressure to control levels was not immediate (2-3 min), as it was with intravenous administration of naloxone. A blockade of the responses by DAME elicited from the vasopressor areas was seen when the animals were treated with naloxone prior to the administration of DAME.
ef al.
sites. In this group of animals (n = 7). saline was injected as a control into the vasopressor sites previously identified with L-glutamate (333 ngsite) and the carotid occlusion response was studied. The blood pressure, heart rate or carotid occlusion response was not modified by injection of saline into these sites (Fig. 4A), whereas a significant decrease in blood pressure, heart rate and carotid occlusion response was observed subsequent to the injection ol DAME into the vasopressor area (Fig. 4B). In the 7 animals studied, a 67”,, attenuation of the carotid occlusion response was observed after the administration of DAME (Fig. 4C). Administration of naloxone (N = 4) reversed the efrect of DAME on the carotid occlusion response. Control carotid occlusion response in these animals was 30 + 4 mmHg. After injection of DAME (50 ngjsite) the carotid occlusion response was reduced to I I + 2 mmHg and complete occlusion recovery of the carotid response (31 _t 4 mmrIg) was seen after the administration of naloxone (I mgikg, i.v.). To rule out the possibility that the attentuation 01 the carotid occlusion response was due to hemodynamic changes, the following experiment was conducted. First control carotid occlusion was performed; 50ngisite of DAME was then injected into the vasopressor area. A fall in blood pressure was elicited by DAME. At the maximal reduction of blood pressure. phenylephrine (I mg,‘ml solution) was infused via the femoral vein. The rate of flow was adjusted to bring the blood pressure back to control and maintain it at that level. Carotid occlusion wax repeated and was found to be attenuated. showing that DAME, and not the marked reduction of blood pressure it produced, was responsible for the attenuation in the carotid occlusion response. Phenylephrine, infused by itself. did not affect the carotid occlusion response.
In this study (,Y = 4) DAME was injected in the with sites, previously identified vasopressor L-glutamate (333 nglsite). The control blood pressure and heart rate in these animals was 83 + IO mmHg and 450 _t 20 bpm, respectively. After the administration of DAME (50 ng;site) the blood pressure decreased 46 k 2 mmHg in the decerebrate preparation (Fig. 5) and this was not significantly different from the decrease in blood pressure (48 i 2 mmHg). elicited with the same dose of DAME in the urethaneanesthetized preparation (N = 5. Fig. ?A).
DIS(‘USSION
In order to study the role of opiate receptors in the ventrolateral medulla, on the reflex regulation of blood pressure, the response to carotid occlusion (COR) was observed before and after the administration of DAME (50ng/site) into the vasopressor
In the present study, L-glutamate was injected to identify a pressor area in the rostra1 ventrolateral medulla. Stimulation of opiate receptors in this area by an analogue of enkephalin. [D-ala’-met’lenkephalinamide (DAME). subsequent to identification
Opiate
(A)
Naloxone
action
in medullary
pressor
area
943
i.v
*‘Pi!!! Nal(lmg/kg
(El 1 Naloxone
l DAME right6Ongl
i.v>
mlcroln)ectlon
l
l left65Ong)
Nal
Rt (2pg)
4
MN
,
Lt (2PQ)
Fig. 3. In each panel, tracings from top to bottom are of mean arterial pressure (MAP), pulse pressure (BP) and heart rate (HR). The specificity of the opiate response after the injection of DAME into the right (Rt) and left (Lt) pressor area was tested using the opiate antagonist naloxone (Nal) HCI. Panel A shows the reversal of the response to DAME (50ng/site) by intravenous administration of naloxone (1 mg/kg) at the points indicated by the arrows. The reversal was immediate. Panel B shows the decrease in blood pressure and heart rate after the injection of DAME (indicated by the arrows) and the reversal of the response following the injection of naloxone (2 pg/site) into the same sites. The reversal of heart rate and blood pressure to control levels is gradual (2-3 min).
by L-glutamate produced a fall in blood pressure and heart rate. The ventrolateral vasopressor area includes the lateral border of the nucleus reticularis (nucl. ret.) gigantocellularis, the medial aspect of the nucleus reticularis parvocellularis and the dorsal portion of the nucleus reticularis lateralis, and is located 1.5-2.2 mm rostra1 to the obex, 1.4-1.8 mm lateral to the basilar artery, and 0.8-1.3 mm from the ventral surface of the medulla (Willette et al., 1983a). Anat-
omically, this area corresponds to the rostra1 C, area of the ventrolateral medulla. Neurons from this area have been shown to project to the thoracic intermediolateral cell column of the spinal cord (Blessing, Goodchild, Dampney and Chalmers, 1981; Ross, Armstrong, Ruggiero, Pickel, Joh and Reis, 1981). Electrical stimulation (Dampney, 1981) and injection of L-glutamate into this area have been shown to produce large pressor responses and bilateral lesion
944 (A)
Saline
Saline 0
DAME
r T i
60
80
a m
c
co
0. I
I 1 MIN
I
I 1 MIN
Fig. 4. Sensitivity ofthe carotid occlusion response (COR) before and after the Injection of DAME (50 ng) into the ventrolateral vasopressor area. The effect of DAME (panel B) on the carotid occlusion (C.O.) is significantly smaller than the effect of the saline (panel A). Panel C graphically illustrates the attenuation of the carotid occlusion response after the injection of DAME. A 67”,, attenuation in the carotid occlusion response is seen after the administrdtion of DAME (hatched bar). No significant difference was observed after the injection of saline (open bar) (n = 7).
of the same area results in a dramatic fall of resting arterial pressure. These studies suggest that this area plays a role in generating and maintaining vasomotor tone. Uhl et al. (1979) demonstrated the presence of enkephalin-like immunoreactivity in the medullary reticular formation. corresponding to the pressor area in the present study. Injections of DAME were, therefore, used to study the effects of activation of opiate receptors in this area on cardiovascular function. The injections of DAME into the ventrolateral medulla caused a fall in blood pressure and heart rate which was dose-related. The inhibitory action of DAME was found to be similar to that caused by lesioning the area electrically, as both produced a fall in blood pressure. Excitation of the ventrolateral medulla with L-glutamate elicited a pressor response. mediated via the sympathetic nervous system because phentolamine, an alpha-adrenergic blocker, abolished the pressor responses. The inhibition of this area might produce a sympathetic withdrawal. causing the fall in blood pressure. The specificity 01 cardiovascular responses to DAME injected in the ventrolateral medulla was tested by administering the opiate antagonist naloxone, both intravenously and by injection. A blockade and reversal of the response of DAME by naloxone suggests that the fall in blood
pressure and heart rate induced by DAME were opiate-receptor specific. The fall in blood pressure induced by the injection of DAME into the pressor sites was immediately reversed to control levels by the intravenous administration of naloxone; blood-borne naloxone may be reaching a greater population ol vasopressor neurons simultaneously. On the other hand. the fall in blood pressure induced by injection of DAME into the pressor sites was reversed gradually (a delay of about 2-3 min) to control levels by the injection of naloxone into the pressor sites: this delay may be explained by the gradual diffusion ol naloxone from the injection site 50 pm (diameter) to the rest of the neurons in the ventrolateral medulla. The attenuation of the response to carotid occlusion after the administration of DAME into the ventrolateral medulla indicates that this area plays II role in the processing of baroreceptor information and that the opiate receptors in this area can modify the reflex regulation of blood pressure and heart rate. The reflex increase in heart rate. which is expected due to the fall in blood pressure, was not observed after the injection of DAME into the ventrolateral medulla, further suggesting that opiates may impair baroreflex mechanisms. Similar attenuation of the baroreceptor reflex after intracerebroventricular administration of DAME has been reported by other\
Opiate
action
in medullary
Decerebrote
pressor
945
area
preporotlon
200 r
a
2 0
I
v
600
200
r
k OL
4 DAME
Rt l50flgl
4
L
I 1 MIN
Li 150ng)
Fig. 5. The injection of DAME (50 ng) into the right (Rt) and left (Lt) ventrolateral vasopressor area (indicated by the arrows) in a midcollicular decerebrate rat showed no significant difference from the responses to DAME elicited in urethane-anesthetized animals shown in Fig. IB. The tracings from top to bottom are mean arterial pressure (MAP), heart rate (HR) and pulse pressure (BP).
(Yukimura, Stock, Stumpf, Unger and Ganten, 1981). This attenuation may be expected because the pressor response during carotid occlusion may involve the neuronal pool in the ventrolateral medulla; inhibition of these neurons by opiates would therefore, attenuate the pressor responses. No significant differences in the action of DAME in decerebrate and urethane-anesthetized animals were observed. These results suggest that: (i) anesthesia did not modify the effects of DAME, and (ii) the cardiovascular effects elicited by injections of DAME into the ventrolateral medulla were not dependent on higher centers, such as the hypothalamus. There are many endogenous substances that have been implicated in the regulation of cardiovascular function in the ventrolateral pressor area. For example, receptors for p-aminobutyric acid (GABA) have been demonstrated in this area by the injection of muscimol, a potent analogue of GABA. Muscimol, like DAME, depressed the neurons in this area, causing a fall in blood pressure (Willette, Krieger, Barcas and Sapru, 1983b). In the present study, atropine sulfate was also injected into the pressor area; administration of this muscarinic antagonist produced a significant decrease in blood pres-
sure suggesting tonic, cholinergic activity in the ventrolateral medulla to generate the vasomotor tone. Centrally, acetylcholine is known to be an excitatory neurotransmitter (Brezenoff and Giuliano, 1982). The present study provides evidence for the pharmacological importance of enkephahns and opiate receptors, which have been reported (Uhl et ul., 1979) to be present in the ventrolateral medulla, the site where cardiovascular neuron pools are located. To summarize, the neurons in the rostra1 ventrolateral medulla, when stimulated with L-glutamate, increase the blood pressure and are inhibited by stimulation of opiate receptors. This area plays a role in the maintenance of vasomotor tone and in reflex cardiovascular regulation. Acknowledgements-This work was supported by grants from the following sources: N.I.H. (HL24347): N.I.H. biomedical research grant (RR05393); American Heart Association (New Jersey); East Orange Veterans Administration Medical Center. REFERENCES
Alexander R. S. (1946) medullary sympathetic rophysiol.9: 205-217.
Tonic and reflex functions of cardiovascular centers. J. Neu-
946
SUSAN
PUNNEN cti a/.
Blessing W. W._ Goodc~~~d A. K., ~ampney R. A. and Chalmers J. P. (1981) Cell groups in the lower brain stem of the rabbit projecting to the spinal cord with special reference to catecholamine-cont~j~ing neurons. Brain Res. 221: 35-55. Bolme P., Fuxe K., Agnati L. F., Bradley R. and Smythies 9. (1978) Cardiovascular effects of morphine and opioid peptides following intracisternal administration in Eur. J. Phurmuc. 48: chloralose-anesthetized rats. 319-324. Brezenoff H. E. and Giuliano R. (1982) Cardiovascular control by cholinergic mechanisms in the central nervous system. A. Rer. Pharmac. Tosir*. 2% 34l--381. Calaresu F. R., Faiers A. A. and Mo~ensan G. J. (1975) Central neural regulation of heart and blood vessels in mammals. Prq. ~~~~~~~~~. 5: l-35. Croxton E. E. (I 959) ~~erne~i~~~,~.~i~t~~~~~.~ r&h ,~~~lj~~~~j~~~ if1 ~~~~j~j#e and ~~#~~~~~~[S&ncc~ pp. 235-239. Dover, New York. Dahlstrom A and Fuxe K. (1964) Evidence for the existence of monoamine-containing neurons in the central nervous system. I. Demonstration of monoamines in the cell bodies of brain stem neurons. Acln ph~siol. sccmd. 62 (suppl. 232): l-55. Rampney R. A. L. (1981) Brain stem mechanisms in the control of arterial pressure. Clin. exp. HyxWw 3: X79-391. Feidberg W. (I 976) The ventral surf&e of the brain stem: a scarcely explored region of pharmacological sensitivity. ~~gu~o.s~~e~~eI: 427-441_ Ganten D., Unger T.. Simon W., Schaz K., Sch~ike~s B.. Mann J. F. E., Speck G.. Lang R. and Rascher W. (1981) Central peptidergic st~muiatio~: Focus on c~rdio~dscular actions of angiotensin and opioid peptides. In: Prrspc+ ke.s in Curdiorusrular Rrseurch (Buckley J. P. and Ferrario C. M., Eds). Vol. 6, pp. 265-282. Raven Press. New York. HGkfelt T., Fuxe K., Goldstein M. and Johansson 0. (1973) Evidence for adrenergic neurons in rat brain. Actor ph,*.riol. wand. 89: 286-288. Krinievic K. and Phillips J. W. (1963) lontophoretic studies of neurons in the mammalian cerebral cortex. J. Phj,.sinl.. Lo&. 265: 274-304. Lang R. E., Unger T., Rascher W. and Ganten D. (1983) In: ~u~~d~~~k of‘ Ps~choprrarmacol (Iversen L. L.,
Iversen S. D. and Snyder S. H., Eds), Vol. 16. pp. 307.-349. Plenum Press, New York. Falkovits M. (1981) Ne~ro~ptides and biogenic amines m central cardiovascular control mechanisms. In: Persp~criz~>,s in Cardioruscuiur Research (Buckley J. F. and Ferrario 0, M., Eds), Vol. 6, pp. 73-87. Raven Press. New York. Pert C. B., Pert A., Cheng J. K. and Fang B. T. W. (1970) a Iinamide: A potent, long acting an[u-al&net’]-enkeph algesic. Science 194: 320-332. Ross C. A., Armstrong D. M., Ruggiero D. A., Pickel V. M., Joh T, H. and Reis D. J. (1981) Adrenaline neurons in the rostra] ventrolateral medulla innervate thoracic spinal.cord: A combined i~nmunoc~toch~mical and ~et~~&rade Transport demonstration. ,~~tir~~.~~j.LPII. 25: X7--262. Sapru H. N. and Krieger A. 5. (1978) Procedure iilr decerebr~ti~n of the rat. ~r~~~ R&s. &if. 3: 675-679. Simantov R., Kuhar M. J., Uhl G. R. and Snyder S. H. (1977) Opioid peptide enkephalin: Immu~ohistochemic~l mapping in rat central nervous system. Proc. nutn. Awl. Sci. U.S.A. 74: 2167-2171. Uhl G. R.. Goodman R. R., Kuhar M. J., Childers S, R. and Snyder S. H. (1979) Immunohistochemical mapping of enkephalin containing cell bodies, fibers and nerve terminals in the brain of the rat. Brain Rrs. 166: 75-94. Wennergren G. and Oberg B. (I 980) Cardiovascular eff’ects elicited from the ventral surface of medulla oblongata in the cat. ~~~~~r.~ ilrcfi. Eur. J. Phwiol. 387: 189-195. Willette R, N., Barcas P. P.. Krieger A. .I. and Sapru H. N, (1983a) Vasoprexsor and depressor area in the rat medulla: ~de~ti~cat~on by ~-glutamate rnicro;~J~t~ons. Nc”I(r~~~l~r~~c~~~#~~ 22t 107 1- 1079. Willette R. N., Punnen S., Krieger A. J. and Sapru H. N. (1984) Hypertensive response following opiate receptor stimulation in the caudal ventrolateral medulla. A’purrrpharmacology 23: 40 i-406. Willette R. N., Krieger A. J., Barcas F. P. and Sapru H. N. (1983b) Medullary y-aminobutyric acid (GABA) receptors and the regulation of blood pressure in the rat. .I. Pharmuc. exp, Thu. 226: 893-899. Yukimura T.. Stock G., Stumpf H., Unger T. and Ganten D. (1981) Effects of [a-ala’lmethionine-enkephalin on blood pressure, heart rate, and baroreceptor reflex sensitivity in ~~ns~i~us cd&. ~~~~~r~~~~~~ 3: 528-533.
pp. 939-946.
1984
Printed in Great Britain. All rights reserved
CARDIOVASCULAR RESPONSE TO INJECTIONS ENKEPHALIN IN THE PRESSBR AREA OF THE VENTROLATERAL MEDULLA*
OF
SUSAN PUNNEN, R. WILLETTE, A. J. KRIECER and H. N. SAPRUT Department of Pharmacology and Section of Neurosurgery, University of Medicine and Dentistry of New Jersey. New Jersey Medical School, Newark, NJ 07103 and V.A. Medical Center, East Orange, NJ 07109, U.S.A.
Summary-The
cardiovascular effects of the injection of an e~kephalin analogue, [D-ala’-mets]enkephalinamide (DAME) into the pressor area of the rostra1 ventrolateral medulla were studied in urethane-anesthetized and decerebrate rats. The excitatory amino acid L-glutamate was used to identify the ventrolateral medulla. The pressor responses to t-glutamate were elicited from an area that included the nucleus reticularis gigantocellularis, the medial aspect of the nucleus reticularis parvocellularis and the dorsal portion of the nucleus reticularis lateralis. Injection (0.1 ~1 volume) of DAME (2.5-500.0 ng/site) into the ventrolateral medulla elicited a dose-related decrease in arterial blood pressure and heart rate and attenuated the carotid occlusion response (COR), Control injections (0.1-0.2~1 vol) of saline into the same area failed to produce any response. The specificity of this opiate response was tested with naloxone HCI, an opiate antagonist, which prevented, as well as reversed, the action of DAME both by intravenous (i.v.) administration and by injection into the ventro~atera~ medulla. It was concluded that the ventrolateral rn~~~~a plays a role in the generation of vasomotor tone and that stimulation of opiate receptors in this area by an e~kepha~in analogue produced hypote~sion, bradycar~~a and modi~~at~on of cardiovascular reflexes. Key words: bradycardia, carotid occlusion, enkephalin, vasotiressor neurons; ventrolateral medulla.
~ypot~nsio~, naloxone, opiate receptors.
and Rascher, 1981; Bolme, Fuxe, Agnati, Bradley and Smythies, 1978; Lang, Unger, Rascher and Ganten, 1983; Palkovits, 198 1). Among them, opioids have been shown to exert potent effects on blood pressure and they are receiving increasing attention since they have been found to be present in the cardiovascular centers of the brain (Sima~t~v, Kuhar, Uhl and Snyder, 1977; Uhl, Goodman, Kuhar, er, 1979). The effect of opiates on cardiovascular regulation in the ventrolateral vasodepressor area, i.e. the A,, area (Dahlstrom and Fuxe, 1964), was studied by Willette, Punnen, Krieger and Sapru (1984). The present study was extended to explore the significance of opiate receptor stimulation in the ventrolateral vasopressor neuron pool, which is located rostra1 to the vasodepressor area and seems to coincide with rostra! C,, epinephrine-containing neurons (H&kfelt, Fuxe, Goldstein and Johansso~, 1973). ~~-a~a~-met5~~~kepha~~~arn~de (~AM~)~ a stable analogue of enkep~alin (Pert, Pert, Cheng and Fang, 1976) was chosen for the study of the effects of opiates on blood pressure, heart rate and cardiovascular reflexes in the rostra1 ventrolateral vasopressor area.
It has become increasingly clear that the functional maintenance of cardiovascular homeostasis is dependent upon the integrity of several nuclei which are located in the medulla (Calaresu, Faiers and Mogenson, 1975). More recently the ventral medulla has been shown to play an important role in the central regulation of blood pressure (Feldberg, 1974; ~arnp~~y~ 1981; Wen~~rgr~n and Oberg, 1980). The presence of depressor and pressor areas in the medulla, regulating sympathetic activity, was demonstrated by Alexander (1946) employing electrical stimulation techniques. Since electrical stimulation evokes responses from cell bodies, fibers of passage the excitatory amino acid and nerve endings, L-glutamate was used to map out the depressor and pressor neuron pools in the ventrolateral medulla, by Krieger and Sapru (1983). Willette, Barcas, L-Glutamate is known to stimulate cell bodies and not axons of passage (Krinjevic and Phillips, 1963). Several peptides from brain have been imprecated in the central regulation of blood pressure (Ganten, Unger, Simon, Schaz, Schiilkens, Mann, Speck, Lang
*A preliminary report of this study has appeared as an abstract in Fedn hoc. Fedn Am. Sum exp. Biol. 42: 653,
METHODS
SuvgicaEprocedures
1983.
tAddress all correspondence to: Hreday N. Sapru, Ph.D., Neurosurgery, Medical Sciences Bldg, H-592, New Jersey Medical School. Newark, NJ 07103, U.S.A. 939
All experiments were carried out acutely in male Wistar rats (Royal Hart, New York) weighing be-
940
SUSAN PUNNEN at d.
tween 250 and 350 g. In the anesthetized preparation urethane (I .O- 1.2 g/kg, i.p.) was used. Experiments were also done using unanesthetized decerebrate animals, as described in detail elsewhere (Sapru and Krieger, 197X). In all experiments the trachea, as well as the femoral vein and artery. was cannulated. The animals were then placed in a stereotaxic instrument (David Kopf Instruments) in a supine position with the upper incisor bar at the level of the interaural line. The upper trachea, larynx. esophagus and surrounding musculature were excised and the longus capitis muscles were removed bilaterally, exposing the occipital foramen and the basal portion of the occipital bone. The basal portion of the occipital bone was then carefully removed. creating a ventral window approx. 6 mm wide and 7 mm long (Willette et al., 1983a). The rostrocaudal, mediolateral and dorsoventral stereotaxic coordinates were in reference to the most caudal aspect of the occipital foramen. basilar artery and ventral surface of the respectively. Upon histological exammedulla, ination. the rostrocaudal landmark was found to be 2.0-2.5 mm caudal to the obex. In all experiments, blood pressure (BP), mean arterial pressure (MAP) and heart rate (HR) were monitored. Blood pressure was recorded from the left femoral artery, using a pressure transducer (Statham P23 Db) and heart rate was monitored by a tachograph (Grass Model 7P4). The rectal temperature was monitored by a digital thermometer (BAT-X, Bailey Instruments) via a rectal probe and maintained at 37 k 0.5 C. Animals were artificially ventilated with room air, using a rodent respirator (Harvard, 680). to avoid secondary cardiovascular effects. In the animals in which the responses to carotid occlusion were elicited. the common carotid arteries were freed from the vagus and sympathetic nerves and the surrounding connective tissue. Carotid occlusion was performed with hemoclips for I5 set intervals before and after the administration of saline and DAME.
L-Glutamate monosodium (L-glu), [o-ala’-met5]enkephalinamide and other drugs used in this study were dissolved in 0.9”,, NaCl (pH 7.0) and injected in a volume of 0.1 PI over a 5 set interval. The solutions of drug were delivered through glass multibarrel micropipettes (Medical Systems, Inc., New York) pulled to an outside tip diameter of approx. 501tm. Unused barrels of the micropipette were filled with 0.9”,,, NaCl and sealed. The vasopressor areas were consistently identified in the ventrolateral medulla by injecting the excitatory amino acid L-glutamate (333 ng:‘site) into an area extending from I.5 to 2.2 mm rostra1 to the obex. I .4 to I .X mm lateral to the basilar artery and 0.X to I .3 mm from the ventral surface. At the end of each experiment, the stereotaxic coordinates of the sites of injection were noted and an
electrolytic lesion was then made by passing a constant current of 3.5 mA for 30 set through a platinum-iridium microelectrode (20 pm O.D.). The brain was then fixed by intravascular perfusion with IO”, formaldehyde. Thin coronal sections ( 12pm) ol the appropriate areas were cut, mounted on microscope slides and stained with cresyl violet for histological identification of the sites of injection. Stutislical
anal~xis
Each animal served as its own control and a paired two-tailed r-test was used to determine whether the mean from a population of differences was zero (Croxton, 1959). Independent f-test was used to test the significance of differences. if any, between the cardiovascular responses, in decerebrate and in urethane-anesthetized rats. Differences were considered significant at P < 0.05. All values are expressed as the mean k the standard error of the mean (SEM). RESULTS
The vasopressor sites in the ventrolateral medulla were identified (Fig. IA) bilaterally by injecting L-glutamate (333 ngjsite). L-Glutamate produced an increase in blood pressure and heart rate. When DAME (50 ng/site) was injected into these sites, the most striking effect was a decrease in blood pressure. Decreases in pulse pressure and heart rate were also observed. The onset of action of this response was immediate (Fig. IB). The histological sites from which DAME elicited the depressor response are shown in Fig. IC. Earlier studies have shown that the vasopressor sites were located in the lateral border of the nucleus reticularis gigantocellularis, the medial aspect of the nucleus reticularis parvocellularis and the dorsal portion of the nucleus reticularis lateralis (Willette et cd., 1983a). After identification of the vasopressor sites with L-glutamate several doses of DAME were injected into these sites to study dose--response relationships. Since tachyphylaxis to repeated administration of DAME was evident. different doses were administered at intervals of 45 min. A dose range of 2.5 to 500.0 ngjsite of DAME was used. The duration and magnitude of the responses in blood pressure and heart rate to DAME were found to be dose-related. A total of 5 animals was used in this study. The control blood pressure and heart rate in the animals was 92 k 4 mmHg and 3X7 + I4 bpm. respectively. Figure 2A shows the semilogarithmic dose-response curve for changes in blood pressure with injections 01 DAME. The fall in blood pressure was I5 + 2 mmHg following 2.5 ng:‘site and, 49 + 3 mmHg a1 lOOng/site. The peak etfect on the blood pressure response was seen at the dose of 100 ng/site; increasing the dose to 500 ng/site did not elicit a furthei decrease in blood pressure. The change in heart rate
Opiate action in medullary pressor area PRESSOR (A)
941
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Fig. 1. In each panel, tracings from top to bottom are of mean arterial pressure (MAP). heart rate (HR), and pulse pressure (BP). The volume of injection of L-glutamate (L-glu, 333 ng) and DAME (50 ng) was 0. I pl. Panel A: typical L-glutamate induced rise in blood pressure and heart rate, after injection into right and left side of the ventrolateral medulla. Panel B: decrease in blood pressure and heart rate following the bilateral injection of DAME into the same sites indicated by the arrows. Control injections of 0. I to 0.2~11of saline produced no responses. Panel C: filled circles indicate the medullary site which elicited the responses of L-glutamate and DAME. Abbreviations: na, nucleus ambiguus; ngc, nucleus gigantocellularis; nrl, nucleus reticularis lateralis; nts, nucleus trdctus solitarius; ntv, nucleus tractus spinalis nervi trigemini; p. pyramidal tract; rpc, nucleus reticularis parvocehukdris.
with DAME was less dramatic than the blood pressure response. As shown in Fig. 2B, at 2.5 ng/site a decrease of 20 + 4 bpm was observed and a maximal decrease of 40 + 5 beats was seen at 100 ng/site of DAME. The duration of action of DAME on blood pressure ranged from 6 to 8 min at 50ng/site to a more prolonged action of I5 to 20 min at 500 ngjsite. To show that changes in blood pressure and heart rate associated with the injection of DAME into the vasopressor area were opiate specific, naloxone, an was administered both intraopiate antagonist, venously (I mg/kg, iv.) and by injection (2 pg/site) into the vasopressor sites. A total of 7 animals was used in this study. The control blood pressure and
heart rate in the animals was 92 + 2 mmHg and 417 + 19 bpm, respectively. After injection of DAME (50ng/site) the fall in blood pressure was 46 f 4mmHg and the heart rate decreased 34 + 3 bpm. Reversal of the cardiovascular effects of DAME to control levels was observed at both routes of administration (i.v. and injection) of naloxone which, by itself, has no effect on blood pressure and heart rate. Figure 3A shows the effect of DAME on blood pressure and heart rate and its reversal by naloxone. Control mean arterial blood pressure (MAP) was 85mmHg and decreased to 35mmHg after bilateral administration of DAME (50 ngjsite), a decrease of 50mmHg; the heart rate (control
942
SUSAN PUNNEN
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Fig. 2. Dose-response curves for depressor and bradycardic responses. Panel A: depressor responses after the bilateral injection of DAME into the ventrolateral vasopressor area. Doses ranging from 2.5 to 500.0 ng were used. Maximal responses were seen with the 100 ng/site. Panel B: decrease in heart rate (HR) following injection of DAME into the ventrolateral medulla at the various doses. All values are expressed as mean + SEM (n = 5).
430 bpm) decreased by 20 bpm. Naloxone (1 mg/kg. i.v.) given 7 min after the administration of DAME immediately reversed blood pressure to control levels. Injection of naloxone into the same vasopressor sites at the peak effect of DAME on blood pressure (60 mmHg decrease) reversed the blood pressure response (Fig. 3B). The time course of the reversal of blood pressure to control levels was not immediate (2-3 min), as it was with intravenous administration of naloxone. A blockade of the responses by DAME elicited from the vasopressor areas was seen when the animals were treated with naloxone prior to the administration of DAME.
ef al.
sites. In this group of animals (n = 7). saline was injected as a control into the vasopressor sites previously identified with L-glutamate (333 ngsite) and the carotid occlusion response was studied. The blood pressure, heart rate or carotid occlusion response was not modified by injection of saline into these sites (Fig. 4A), whereas a significant decrease in blood pressure, heart rate and carotid occlusion response was observed subsequent to the injection ol DAME into the vasopressor area (Fig. 4B). In the 7 animals studied, a 67”,, attenuation of the carotid occlusion response was observed after the administration of DAME (Fig. 4C). Administration of naloxone (N = 4) reversed the efrect of DAME on the carotid occlusion response. Control carotid occlusion response in these animals was 30 + 4 mmHg. After injection of DAME (50 ngjsite) the carotid occlusion response was reduced to I I + 2 mmHg and complete occlusion recovery of the carotid response (31 _t 4 mmrIg) was seen after the administration of naloxone (I mgikg, i.v.). To rule out the possibility that the attentuation 01 the carotid occlusion response was due to hemodynamic changes, the following experiment was conducted. First control carotid occlusion was performed; 50ngisite of DAME was then injected into the vasopressor area. A fall in blood pressure was elicited by DAME. At the maximal reduction of blood pressure. phenylephrine (I mg,‘ml solution) was infused via the femoral vein. The rate of flow was adjusted to bring the blood pressure back to control and maintain it at that level. Carotid occlusion wax repeated and was found to be attenuated. showing that DAME, and not the marked reduction of blood pressure it produced, was responsible for the attenuation in the carotid occlusion response. Phenylephrine, infused by itself. did not affect the carotid occlusion response.
In this study (,Y = 4) DAME was injected in the with sites, previously identified vasopressor L-glutamate (333 nglsite). The control blood pressure and heart rate in these animals was 83 + IO mmHg and 450 _t 20 bpm, respectively. After the administration of DAME (50 ng;site) the blood pressure decreased 46 k 2 mmHg in the decerebrate preparation (Fig. 5) and this was not significantly different from the decrease in blood pressure (48 i 2 mmHg). elicited with the same dose of DAME in the urethaneanesthetized preparation (N = 5. Fig. ?A).
DIS(‘USSION
In order to study the role of opiate receptors in the ventrolateral medulla, on the reflex regulation of blood pressure, the response to carotid occlusion (COR) was observed before and after the administration of DAME (50ng/site) into the vasopressor
In the present study, L-glutamate was injected to identify a pressor area in the rostra1 ventrolateral medulla. Stimulation of opiate receptors in this area by an analogue of enkephalin. [D-ala’-met’lenkephalinamide (DAME). subsequent to identification
Opiate
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943
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Fig. 3. In each panel, tracings from top to bottom are of mean arterial pressure (MAP), pulse pressure (BP) and heart rate (HR). The specificity of the opiate response after the injection of DAME into the right (Rt) and left (Lt) pressor area was tested using the opiate antagonist naloxone (Nal) HCI. Panel A shows the reversal of the response to DAME (50ng/site) by intravenous administration of naloxone (1 mg/kg) at the points indicated by the arrows. The reversal was immediate. Panel B shows the decrease in blood pressure and heart rate after the injection of DAME (indicated by the arrows) and the reversal of the response following the injection of naloxone (2 pg/site) into the same sites. The reversal of heart rate and blood pressure to control levels is gradual (2-3 min).
by L-glutamate produced a fall in blood pressure and heart rate. The ventrolateral vasopressor area includes the lateral border of the nucleus reticularis (nucl. ret.) gigantocellularis, the medial aspect of the nucleus reticularis parvocellularis and the dorsal portion of the nucleus reticularis lateralis, and is located 1.5-2.2 mm rostra1 to the obex, 1.4-1.8 mm lateral to the basilar artery, and 0.8-1.3 mm from the ventral surface of the medulla (Willette et al., 1983a). Anat-
omically, this area corresponds to the rostra1 C, area of the ventrolateral medulla. Neurons from this area have been shown to project to the thoracic intermediolateral cell column of the spinal cord (Blessing, Goodchild, Dampney and Chalmers, 1981; Ross, Armstrong, Ruggiero, Pickel, Joh and Reis, 1981). Electrical stimulation (Dampney, 1981) and injection of L-glutamate into this area have been shown to produce large pressor responses and bilateral lesion
944 (A)
Saline
Saline 0
DAME
r T i
60
80
a m
c
co
0. I
I 1 MIN
I
I 1 MIN
Fig. 4. Sensitivity ofthe carotid occlusion response (COR) before and after the Injection of DAME (50 ng) into the ventrolateral vasopressor area. The effect of DAME (panel B) on the carotid occlusion (C.O.) is significantly smaller than the effect of the saline (panel A). Panel C graphically illustrates the attenuation of the carotid occlusion response after the injection of DAME. A 67”,, attenuation in the carotid occlusion response is seen after the administrdtion of DAME (hatched bar). No significant difference was observed after the injection of saline (open bar) (n = 7).
of the same area results in a dramatic fall of resting arterial pressure. These studies suggest that this area plays a role in generating and maintaining vasomotor tone. Uhl et al. (1979) demonstrated the presence of enkephalin-like immunoreactivity in the medullary reticular formation. corresponding to the pressor area in the present study. Injections of DAME were, therefore, used to study the effects of activation of opiate receptors in this area on cardiovascular function. The injections of DAME into the ventrolateral medulla caused a fall in blood pressure and heart rate which was dose-related. The inhibitory action of DAME was found to be similar to that caused by lesioning the area electrically, as both produced a fall in blood pressure. Excitation of the ventrolateral medulla with L-glutamate elicited a pressor response. mediated via the sympathetic nervous system because phentolamine, an alpha-adrenergic blocker, abolished the pressor responses. The inhibition of this area might produce a sympathetic withdrawal. causing the fall in blood pressure. The specificity 01 cardiovascular responses to DAME injected in the ventrolateral medulla was tested by administering the opiate antagonist naloxone, both intravenously and by injection. A blockade and reversal of the response of DAME by naloxone suggests that the fall in blood
pressure and heart rate induced by DAME were opiate-receptor specific. The fall in blood pressure induced by the injection of DAME into the pressor sites was immediately reversed to control levels by the intravenous administration of naloxone; blood-borne naloxone may be reaching a greater population ol vasopressor neurons simultaneously. On the other hand. the fall in blood pressure induced by injection of DAME into the pressor sites was reversed gradually (a delay of about 2-3 min) to control levels by the injection of naloxone into the pressor sites: this delay may be explained by the gradual diffusion ol naloxone from the injection site 50 pm (diameter) to the rest of the neurons in the ventrolateral medulla. The attenuation of the response to carotid occlusion after the administration of DAME into the ventrolateral medulla indicates that this area plays II role in the processing of baroreceptor information and that the opiate receptors in this area can modify the reflex regulation of blood pressure and heart rate. The reflex increase in heart rate. which is expected due to the fall in blood pressure, was not observed after the injection of DAME into the ventrolateral medulla, further suggesting that opiates may impair baroreflex mechanisms. Similar attenuation of the baroreceptor reflex after intracerebroventricular administration of DAME has been reported by other\
Opiate
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Fig. 5. The injection of DAME (50 ng) into the right (Rt) and left (Lt) ventrolateral vasopressor area (indicated by the arrows) in a midcollicular decerebrate rat showed no significant difference from the responses to DAME elicited in urethane-anesthetized animals shown in Fig. IB. The tracings from top to bottom are mean arterial pressure (MAP), heart rate (HR) and pulse pressure (BP).
(Yukimura, Stock, Stumpf, Unger and Ganten, 1981). This attenuation may be expected because the pressor response during carotid occlusion may involve the neuronal pool in the ventrolateral medulla; inhibition of these neurons by opiates would therefore, attenuate the pressor responses. No significant differences in the action of DAME in decerebrate and urethane-anesthetized animals were observed. These results suggest that: (i) anesthesia did not modify the effects of DAME, and (ii) the cardiovascular effects elicited by injections of DAME into the ventrolateral medulla were not dependent on higher centers, such as the hypothalamus. There are many endogenous substances that have been implicated in the regulation of cardiovascular function in the ventrolateral pressor area. For example, receptors for p-aminobutyric acid (GABA) have been demonstrated in this area by the injection of muscimol, a potent analogue of GABA. Muscimol, like DAME, depressed the neurons in this area, causing a fall in blood pressure (Willette, Krieger, Barcas and Sapru, 1983b). In the present study, atropine sulfate was also injected into the pressor area; administration of this muscarinic antagonist produced a significant decrease in blood pres-
sure suggesting tonic, cholinergic activity in the ventrolateral medulla to generate the vasomotor tone. Centrally, acetylcholine is known to be an excitatory neurotransmitter (Brezenoff and Giuliano, 1982). The present study provides evidence for the pharmacological importance of enkephahns and opiate receptors, which have been reported (Uhl et ul., 1979) to be present in the ventrolateral medulla, the site where cardiovascular neuron pools are located. To summarize, the neurons in the rostra1 ventrolateral medulla, when stimulated with L-glutamate, increase the blood pressure and are inhibited by stimulation of opiate receptors. This area plays a role in the maintenance of vasomotor tone and in reflex cardiovascular regulation. Acknowledgements-This work was supported by grants from the following sources: N.I.H. (HL24347): N.I.H. biomedical research grant (RR05393); American Heart Association (New Jersey); East Orange Veterans Administration Medical Center. REFERENCES
Alexander R. S. (1946) medullary sympathetic rophysiol.9: 205-217.
Tonic and reflex functions of cardiovascular centers. J. Neu-
946
SUSAN
PUNNEN cti a/.
Blessing W. W._ Goodc~~~d A. K., ~ampney R. A. and Chalmers J. P. (1981) Cell groups in the lower brain stem of the rabbit projecting to the spinal cord with special reference to catecholamine-cont~j~ing neurons. Brain Res. 221: 35-55. Bolme P., Fuxe K., Agnati L. F., Bradley R. and Smythies 9. (1978) Cardiovascular effects of morphine and opioid peptides following intracisternal administration in Eur. J. Phurmuc. 48: chloralose-anesthetized rats. 319-324. Brezenoff H. E. and Giuliano R. (1982) Cardiovascular control by cholinergic mechanisms in the central nervous system. A. Rer. Pharmac. Tosir*. 2% 34l--381. Calaresu F. R., Faiers A. A. and Mo~ensan G. J. (1975) Central neural regulation of heart and blood vessels in mammals. Prq. ~~~~~~~~~. 5: l-35. Croxton E. E. (I 959) ~~erne~i~~~,~.~i~t~~~~~.~ r&h ,~~~lj~~~~j~~~ if1 ~~~~j~j#e and ~~#~~~~~~[S&ncc~ pp. 235-239. Dover, New York. Dahlstrom A and Fuxe K. (1964) Evidence for the existence of monoamine-containing neurons in the central nervous system. I. Demonstration of monoamines in the cell bodies of brain stem neurons. Acln ph~siol. sccmd. 62 (suppl. 232): l-55. Rampney R. A. L. (1981) Brain stem mechanisms in the control of arterial pressure. Clin. exp. HyxWw 3: X79-391. Feidberg W. (I 976) The ventral surf&e of the brain stem: a scarcely explored region of pharmacological sensitivity. ~~gu~o.s~~e~~eI: 427-441_ Ganten D., Unger T.. Simon W., Schaz K., Sch~ike~s B.. Mann J. F. E., Speck G.. Lang R. and Rascher W. (1981) Central peptidergic st~muiatio~: Focus on c~rdio~dscular actions of angiotensin and opioid peptides. In: Prrspc+ ke.s in Curdiorusrular Rrseurch (Buckley J. P. and Ferrario C. M., Eds). Vol. 6, pp. 265-282. Raven Press. New York. HGkfelt T., Fuxe K., Goldstein M. and Johansson 0. (1973) Evidence for adrenergic neurons in rat brain. Actor ph,*.riol. wand. 89: 286-288. Krinievic K. and Phillips J. W. (1963) lontophoretic studies of neurons in the mammalian cerebral cortex. J. Phj,.sinl.. Lo&. 265: 274-304. Lang R. E., Unger T., Rascher W. and Ganten D. (1983) In: ~u~~d~~~k of‘ Ps~choprrarmacol (Iversen L. L.,
Iversen S. D. and Snyder S. H., Eds), Vol. 16. pp. 307.-349. Plenum Press, New York. Falkovits M. (1981) Ne~ro~ptides and biogenic amines m central cardiovascular control mechanisms. In: Persp~criz~>,s in Cardioruscuiur Research (Buckley J. F. and Ferrario 0, M., Eds), Vol. 6, pp. 73-87. Raven Press. New York. Pert C. B., Pert A., Cheng J. K. and Fang B. T. W. (1970) a Iinamide: A potent, long acting an[u-al&net’]-enkeph algesic. Science 194: 320-332. Ross C. A., Armstrong D. M., Ruggiero D. A., Pickel V. M., Joh T, H. and Reis D. J. (1981) Adrenaline neurons in the rostra] ventrolateral medulla innervate thoracic spinal.cord: A combined i~nmunoc~toch~mical and ~et~~&rade Transport demonstration. ,~~tir~~.~~j.LPII. 25: X7--262. Sapru H. N. and Krieger A. 5. (1978) Procedure iilr decerebr~ti~n of the rat. ~r~~~ R&s. &if. 3: 675-679. Simantov R., Kuhar M. J., Uhl G. R. and Snyder S. H. (1977) Opioid peptide enkephalin: Immu~ohistochemic~l mapping in rat central nervous system. Proc. nutn. Awl. Sci. U.S.A. 74: 2167-2171. Uhl G. R.. Goodman R. R., Kuhar M. J., Childers S, R. and Snyder S. H. (1979) Immunohistochemical mapping of enkephalin containing cell bodies, fibers and nerve terminals in the brain of the rat. Brain Rrs. 166: 75-94. Wennergren G. and Oberg B. (I 980) Cardiovascular eff’ects elicited from the ventral surface of medulla oblongata in the cat. ~~~~~r.~ ilrcfi. Eur. J. Phwiol. 387: 189-195. Willette R, N., Barcas P. P.. Krieger A. .I. and Sapru H. N, (1983a) Vasoprexsor and depressor area in the rat medulla: ~de~ti~cat~on by ~-glutamate rnicro;~J~t~ons. Nc”I(r~~~l~r~~c~~~#~~ 22t 107 1- 1079. Willette R. N., Punnen S., Krieger A. J. and Sapru H. N. (1984) Hypertensive response following opiate receptor stimulation in the caudal ventrolateral medulla. A’purrrpharmacology 23: 40 i-406. Willette R. N., Krieger A. J., Barcas F. P. and Sapru H. N. (1983b) Medullary y-aminobutyric acid (GABA) receptors and the regulation of blood pressure in the rat. .I. Pharmuc. exp, Thu. 226: 893-899. Yukimura T.. Stock G., Stumpf H., Unger T. and Ganten D. (1981) Effects of [a-ala’lmethionine-enkephalin on blood pressure, heart rate, and baroreceptor reflex sensitivity in ~~ns~i~us cd&. ~~~~~r~~~~~~ 3: 528-533.