83

Brain Research, 549 (1991) 83-89 © 1991 Elsevier Science Publishers B.V. 0006-8993/91/$03.50 ADONIS 000689939116600H

BRES 16600

NMDA antagonists attenuate hypertension induced by carotid clamping in the rostral ventrolateral medulla of rats Ming-Chieh Kao z, H.K. Lee 2, C.Y. Chai I and Yun Wang 2 1Institute of Biomedical Sciences Academia Sinica, Taipei, Taiwan (R.O.C.) and 2Department of Pharmacology, National Defense Medical Center, Taipei, Taiwan (R. O. C.)

(Accepted 11 January 1991) Key words: Baroreceptor reflex; Excitatory amino acid; N-Methyl-D-aspartate

The purpose of these experiments were to study the interactions of N-methyI-D-aspartate (NMDA) with baroreceptor reflexes induced by transient carotid clamping. Adult male Sprague-Dawley rats were anesthetized with urethane. Bilateral common carotid artery occlusion resulted in a reversible and reproducible hypertension in the vagotomized animals. This hypertensive reaction was blocked by intraventricular injection of NMDA antagonists, such as 2-amino-7-phosphono-heptaneoate (AP-7) and pheneyclidine (PCP). We also found that blood pressure-sensitive neurons of the rostral ventrolateral medulla (RVLM) could be classified into two groups, on the basis of their responses to norepinephrine given intravenously. Using pressure microejection and single unit recording, we observed that clamping of the common carotids resulted in excitation of type I neurons. This evoked excitation, similar to that induced by NMDA, was blocked by locally applied AP-7. However, the carotid occlusion-induced responses of type II neurons were not blocked by AP-7. In conclusion, the present data suggest that NMDA receptors are involved in hypertensive responses during carotid occlusion, perhaps involving a site in the rostral ventrolateral medulla. INTRODUCTION The baroreceptor reflex is one of the primary mechanisms whereby the central nervous system regulates peripheral cardiovascular functions. Clamping both common carotids results in a reflex increase in blood pressure. At least, two regions in the medulla are involved this reflex. The nucleus of the tractus solitarius (NTS) receives primary afferent input from baroreceptors 18, whereas the rostral ventrolateral medulla (RVLM) provides tonic excitatory drive to preganglionic sympathetic neurons 17. Interestingly, both areas are sensitive to exogenous application of excitatory amino acidsS,10,19. Recent studies have shown that excitatory amino acids regulate cardiovascular functions, at least, in the medulla oblongata. N M D A , quisqualate, and kainate applied topically to the ventral medullary surface resulted in an increase in blood pressure 14,16. MK-801, a non-competitive antagonist at N M D A receptors, attenuated the Bezold-Jarisch reflex elicited by 5-hydroxytryptamine 22. We and others have previously observed that homocysteic acid, applied directly to the ventrolateral medulla, produced a hypertensive response in rats and cats 11. On

the other hand, microinjection of L-glutamate or N M D A into caudal ventral medulla produced decreases in arterial pressure 8'1°. These suggested that exogeneously applied excitatory amino acids modulate cardiovascular functions in the medulla. However, whether endogeneous excitatory ligands are involved in these cardiovascular reactions is not clear. It has been reported that hypertension may be induced by noxious stimulation 24. Several monoamine neurotransmitters were suggested to mediate this reaction, such as norepinephrine and serotonin 1'21. N M D A receptors are closely associated with pain pathways. However, the involvement of N M D A receptors in this pain-induced hypertension has not been defined. The present experiments were designed to explore the role of N M D A receptors in reflexly evoked hypertension. We found that both phencyclidine (PCP) and 2-amino7-phosphono-heptaneoate (AP-7) attenuated hypertensive reactions induced by carotid clamping. Furthermore, we found that two groups of neurons recorded in the RVLM showed different responses to norepinephrine and to carotid occlusion. Excitatory responses in one subpopulation were antagonized by N M D A blockers.

Correspondence: Y. Wang, Department of Pharmacology, National Defense Medical Center, P.O. Box 90048-504 Taipei, Taiwan, R.O.C.

84 MATERIALS AND METHODS

RESULTS

Surgical preparation

Interactions o f N M D A with A P - 7 and P C P on arterial pressure

Adult male Sprague-Dawley rats (250-350 g) were anesthetized with urethane (1.25 g/kg, i.p.). The femoral vein and artery were cannulated for drug administration and for monitoring of blood pressure respectively. The animals were intubated to allow spontaneous breathing and placed in a stereotaxic frame. Body temperature was monitored with a thermistor probe and maintained at 37 °C with a heating pad.

In 20 rats studied, injection of N M D A into the left lateral cerebral ventricle produced a dose-related increase in systemic arterial blood pressure (Fig. 1). Doses higher than 300 nmol produced an initial increase of arterial blood pressure up to 80 mmHg, followed by

Interactions of NMDA and its antagonists in the regulation of arterial pressure Drugs were injected into the left cerebral ventricle (1.0 mm left lateral, 0.5 mm posterior to bregma, 4.5 mm below the skull surface) using a Hamilton syringe. NMDA-induced (0.3, 3, 30 nmol, i.c.v.) changes in blood pressure were measured. To evaluate specificity, NMDA antagonist (AP-7 or PCP) was given (300 nmol, i.c.v.) 30-60 s before the NMDA application. Comparisons were made with control experiments, in which 30/tl of 165 mM NaCI was given prior to NMDA.

A.

60 0

,°I

•

NMDA 0,9~ NaCl + NMDA

I !

P ) 0.05

40

•

v

30 an

Effects of AP-7 and PCP on the increase of arterial pressure during clamping of the common carotid arteries

20

The baroreceptor reflex was induced by clamping the common carotids for 100 s. Cervical vagotomy was performed to minimize any local mechanical stimulation. AP-7 or PCP (300 nmol, i.c.v.) was given 30 to 60 s before the clamping.

10

Effects of AP-7 and PCP on arterial pressure during tail pinching

0

B.

A 2-inch paper clip with a pressure of 13.5 + 0.5 kg/cm 2 was applied to the tail, 10 cm from the distal end, for 30 s to produce a noxious stimulus. The change in blood pressure induced by this stimulation was compared before and after 165 mM saline (30/A, i.c.v.), AP-7, or PCP application (300 nmol, i.c.v.).

60

~ 5O 4O

~ :~D0:3~ m o I + N M D A

* P(0.05 ~/~,

~. 30

Electrophysiological effects of NMDA, AP-7 and carotid baroreceptor reflexes on the pressure-sensitive neurons of the rostral ventrolateral medulla (RVLM) Extracellular action potentials were recorded from single neurons of rostral ventrolateral medulla (1.9 mm left lateral, 2.0 mm anterior to the obex, and 2.5 mm below the brain surface) through the 5 M NaCl-filled barrel of 4-barrel micropipettes which were constructed as previously described 23. Action potentials were amplified and filtered. A window discriminator was used to separate signals from background noise. Spontaneous activity was integrated over one second epochs by a ratemeter and displayed on a strip chart recorder. Drugs were applied locally from 3 barrels of the micropipette by pressure microejection. The magnitude of pressure delivered was regulated by a pneumatic pump (1-30 pounds per square inch). Pressure ejection from glass micropipettes has been reported to release the drug in an amount linearly related to pressure and time 4. NMDA and AP-7 were applied directly to the neurons of RVLM. Sites of recording were identified, at the end of experiment, by histological processing after microejection of Fast green directly through one barrel of the multibarrel pipette. Drugs were dissolved in 0.9% saline. Drugs used for pressure microejection were: N-methyl-D-aspartate (5 mM, Sigma); DL2-amino-7-phosphono-heptanic acid (1 raM, Sigma). Data were expressed as mean +-. S.E.M. and analyzed for statistical significance using Student's t-test and paired Student's t-test. A change of 25% or more in the spontaneous firing rate in response to a drug was the criterion to used define whether a neuron was excited or inhibited. Statistical significance of the tabulated data of type I neurons and type II of RVLM was determined using the Chi-square test and Fisher's exact test. Differences were considered to be significant at P < 0.05.

"~ 20 10

Ce 60

"~ 50

O NMDA A PCP 0.3~ m o l + NMDA •

~. m.

.,o

p
0.05, paired Student's t-test). B: 300 nmol of AP-7 (n = 8) or C: PCP significantly antagonized NMDA-induced hypertension (n = 8, p < 0.05). Data were expressed as mean + S.E.M.

85 hypotension and dyspnea. The latter responses may be due to the neurotoxic effects of N M D A 16. The threshold dose of N M D A for increasing arterial pressure was 3 nmol. The increases in arterial pressure began immediately after injection and lasted for 5-15 min. AP-7 alone (300 nmol) produced no change in blood pressure. However, it antagonized NMDA-evoked hypertension (Fig. 1). PCP, a non-competitive antagonist of the N M D A receptor, also blocked NMDA-induced hypertension at a dose of 300 nmol (Fig. 1).

inhibition was reversible. Twenty-five to forty-five min after the application of AP-7, the hypertension induced by carotid clamping had recovered.

Interaction of AP-7 and PCP with pain-induced hypertension In 18 rats studied, tail pinching increased blood pressure by 10 to 15 mmHg. This reaction was not antagonized by AP-7, PCP or saline (Fig. 4, Table I).

Antagonistic effects of AP-7 and PCP on the increase of arterial pressure induced by the baroreceptor reflex

Electrophysiological interactions of NMDA and AP-7 with carotid baroreceptor reflex in the neurons of the rostral ventrolateral medulla

Twenty-six animals were suitable for analysis. Baroreceptor reflex was induced by bilateral carotid clamping. Clamping for 100 s produced an initial hypotensive reaction lasting for 8 s and then an increased arterial blood pressure (Figs. 2,3). Both AP-7 and PCP did not alter the initial hypotensive response induced by carotid clamping. On the other hand, the pressor effect was suppressed by pretreatment i.c.v, with AP-7 or PCP, but not with saline. (Fig. 2, Table I). The AP-7-induced

Two groups of pressure-sensitive neurons of RVLM were identified according by their electrophysiological response to norepinephrine-induced hypertension (20 nmol, i.v.). Type I neurons were inhibited while type II neurons were excited during the hypertensive period. Twenty-nine pressure-sensitive neurons of RVLM from 20 rats were recorded. Of these, 15 neurons were considered to be the type I neurons (Fig. 5A). The other 14 were type II neurons (Fig. 6B). Locally applied

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wF-

~ _ _

Fig. 2. Interactions of saline, AP-7 and PCP with carotid clamping-induced hypertension. Saline (30 /zl, i.c.v.) did not alter the clamping-mediated elevation of systemic and mean arterial pressure (A2 vs A1; A4 vs A3). However, AP-7 and PCP (300 nmol, i.c.v.) antagonized the hypertension induced by carotid clamping (B2 vs B1, C2 vs C1). The duration of PCP's antagonism was shorter than that of AP-7 (B2 vs C2). Horizontal bars under each tracing represent the duration of carotid clamping.

86 TABLE I

T A B L E II

Interactions of AP-7, PCP and saline with hypertension induced by bilateral common carotid arteries clamping or tail-pinching

NM DA- and carotid clamping-induced electrophysiological responses in the type I and II of pressure-sensitive neurons of R VL M

n

Control

After i. c. v. injection

Carotid clamping-induced changes orB. P. (mmHg) Saline AP-7 PCP

6 15 5

43.3 + 10.5 42.6 + 5.4 44.0 + 8.7

37.5 + 8.8 3.0 + 9.2"** -19.0 + 7.6*'**

Tail pinching-induced changes of B.P. (mmHg) Saline AP-7 PCP

5 8 5

13.0 + 4.1 13.1 + 0.9 13.0 + 2.5

14.0 + 4.3 18.7 + 4.0 16.0 + 1.0

n = n u m b e r of animals studied. * C o m p a r e d with control group, P < 0.05. ** C o m p a r e d with saline treatment, P < 0.05.

200 T Iso t !

(~)

Carotid occlusion

•

APT 0.3/~mol +

1607

Type H neurons

15" 15 0 0

14 14 0 0

15" 15 0 0

14 0 14 0

NMDA -induced Excitation antagonized by AP-7 not antagonized by AP-7 Inhibition

Carotid clamping-induced Excitation antagonized by AP-7 not antagonized by AP-7 Inhibition * N u m b e r of neurons.

NMDA excited both type I and type II neurons (Figs. 5B,6A). NMDA-induced excitations were reversibly antagonized by AP-7 (Figs. 5C,6B). Carotid clamping, similar to NMDA, induced neuronal excitation in both

A.

Type 1 neurons

Carotid occlusion

types of neurons (Figs. 5B,6A). Interestingly, only excitation in the type I neurons was blocked by AP-7 (Fig. 5C; Table II). The excitatory response of Type II neurons, however, were weakly antagonized by the locally applied AP-7 (Fig 6B).

A.

L"F

* P ( 0.05

--

2. Saline

i

12o+ m

I00 ~

200

* M.B.P.

0oii

3.

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60 ~40i

- I

I

I

I

I

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B.E

B. z°° T 180+

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Carotid occlusion

•

PCP 0 3 ~mol + Carotid occlusion

3.

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140+ 120 C.

m

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80

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nmalt| 100 50

< 0.05

60 40

2. PCP

1

100

I

I

I

I

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l

0

20

40

60

80

100

Time

( sec. )

Fig. 3. Effects of AP-7 and PCP on m e a n blood pressure after carotid clamping. Clamping of both carotids for 100 s (horizontal bar) produced an initial hypotension and then a hypertensive reaction. A: AP-7 (300 nmol, n = 15 i.c.v.) and B: PCP (300 nmol, n = 5, i.c.v.) antagonized carotid clamping-induced hypertension. However, the initial hypotensive reaction was not affected by AP-7 or PCP. Data were plotted as m e a n + S.E.M.

zoo V

3.

N.B.p. 150 I"- ~

4. j

-

-

mmH! tO0 t

5O

Fig. 4. Interactions of saline, AP-7 and PCP with hypertension induced by tail pinching. Tail pinching delivered through a clip (horizontal bar u n d e r each tracing) produced an elevation of systemic and m e a n blood pressure. Saline (A2 vs A1), AP-7 (300 nmol, i.c.v., B2 vs B1) or P C P (300 nmol, C2 vs C1) did not alter this hypertensive reaction.

87 E.

A. 200 B.P. 150 1oo 5O

//'

eo

clamp

200

B.P. 150 mm~ 100 50

c. ~00 IB.P. 150~mmHg 50 t-

...... ~/Immmmmmm,,,,~

I001-AP7 m

B

D. 200 F B.P. 1501 mmHg 100F

......

50 u.

,

Fig. 5. AP-7 antagonized NMDA- and carotid clamping-induced neuronal excitation in a type I pressure-sensitive neuron in RVLM. The upper and lower traces represent systemic blood pressure and the neuronal activity, respectively. The neuronal activity was inhibited (A, lower trace) by the systemic administration of norepinephrine (20 nmol, i.v.). Microejection of NMDA (8 psi x 5 s) and carotid damping (B, dotted line) increased the neuronal discharge rate (B, lower trace). AP-7 antagonized NMDA- and carotid clamping-induced neuronal excitation (C). The AP-7-mediated antagonism is reversible because 20 min after the AP-7 application, NMDA- or carotid clamping-mediated electrophysiological responses recovered (D). The vertical calibration of the neuronal histogram is in spikes per second. In this and the following ratemeter records, the duration of application of the drug is indicated by solid and dashed lines above the trace. Histological verification of recording site (triangle) is shown in (E). Abbrevations: Nts, nucleus tractus solitarius; NV, nucleus of the trigeminal nerve; Oli, inferior oliv; Py, pyramidal tract; Amb, nucleus ambiguus; mlf, medial longitudinal fasciculus.

DISCUSSION We found that N M D A , applied into the lateral cerebral ventricle, increased systemic arterial blood pressure. This drug-induced pressor reaction was probably mediated by N M D A receptors in the CNS because it was dose-dependent and, furthermore, was blocked by N M D A antagonists, such as AP-7 and PCP, given intraventricularly. It is possible that carotid clamping activates, at least, 3 processes which alter blood pressure. These include ischemia of the forebrain, stimulation of the vagus nerve,

and induction of the baroreceptor reflex. Our previous findings indicate that alternation of blood pressure induced by clamping of carotids for 100 s is little influenced by decerebration and is abolished by denervation of bilateral glossopharyngeal nerves in vagotomized animals. These findings suggest that the changes of blood pressure elicited by clamping for 100 s in the vagotomized animals studied here is primarily mediated through the baroreceptor reflex. Carotid occlusion induced a biphasic response in the vagotomized animals. This was characterized by an initial hypotensive reaction and then a subsequent hypertensive

88

clamp

A

°...°o°o*

200 B,P. mmHg 150 100 50

NE

B B.P. 20 mmHg 15 10 5,,

~ B ~ m m n n m m m n m m m w l m m m m m m m m m m w m m m n n m m s m m n m m m m m m m m ~ n m ~ m ~

Hz

Fig. 6. AP-7 antagonized NMDA- but not carotid occlusion-mediated neuronal excitations a the type II pressure sensitive neuron of RVLM. A: microinjection of NMDA (21 psi x 5 s) or carotid damping (upper trace) produced neuronal excitation. B: AP-7 antagonized NMDA-induced excitation. Carotid clamping-induced excitation, however, was only slightly altered. This neuron was considered to be a type II neuron because it was excited by the systemic application of norepinephrine (20 nmol, i.v.). response. The hypotensive effect was probably mediated via the carotid sinus nerve stimulation induced by stretch while the pressor response was associated with the chemoreceptor or baroreceptor reflexes as indicated above. We found that centrally applied AP-7 did not influence the initial response. This implies that the depressor response was not mediated through N M D A receptors. On the other hand, the hypertensive reaction was attenuated by either AP-7 or PCP. Interestingly, the tail-pinch-induced hypertension was not altered by AP-7. These data suggest that N M D A is specifically involved with baroreceptor-reflex-induced hypertension. We found that pressure-sensitive neurons in the RVLM were excited by local N M D A application and this excitation was antagonized by AP-7. This suggests that these neurons contain N M D A binding sites. Carotid occlusion also resulted in excitation of pressure-sensitive neurons. However, only the excitation of type I neurons was antagonized by AP-7. It is possible that carotid clamping activates two excitatory pathways to pressuresensitive neurons of RVLM, but only type I neurons possess an NMDA-mediated input. Ketamine, which is a derivative of PCP with fewer side effects and shorter duration of action, was considered a

relative safe surgical anesthetic. It has been reported that PCP analogs produce hypertension in animals and man 6. In contrast, we found that PCP antagonized the pressor response induced by N M D A or baroreceptor reflexes. These findings imply that PCP analogs may be harmful to patients with problems of blood pressure regulation. This conjecture is further supported by the clinical findings that ketamine is problematic in hypovolemic patients 12. It has been reported that that NMDA-mediated reactions can be enhanced by glycine 2 through the strychnineinsensitive glycine receptor 9. The glycine-modulated N M D A responses have been found in the forebrain and hippocampus 3"15. However, in preliminary studies, we found that an injection of glycine (0.3-30 nmol) into the lateral ventricle did not potentiate N M D A or carotid clamping-induced hypertension. It was reported that the distribution of strychnine-insensitive glycine binding site in the brainstem was much less than those in the hippocampus, cerebral cortex, caudate, thalamus, and cerebellum 13. Whether the glycine receptors in the RVLM are mainly strychnine-sensitive requires further investigation. In conclusion, the present data demonstrated that N M D A receptors are involved in the hypertensive

89 reaction during Carotid clamping in the rostral ventrolateral medulla. REFERENCES 1 Chipkin, R.E. and Latranyi, M.B., Subplantar yeast injection induces a non-naloxone reversible antinociception in spontaneously hypertensive rats, Brain Research, 303 (1984) 1-6. 2 Cotman, C.W., Monaghan, D.T., Onersen, O.P. and StormMathisen, J., Anatomical organization of excitatory amino acid receptors and their pathways, Trends Neurosci., 10 (1987) 273-280. 3 Cotman, C.W. and Iversen, L.L., Excitatory amino acids in the brain-focus on NMDA receptors, Trends Neurosci., 10 (1987) 263-265. 4 Gerhardt, G.A. and Palmer, M.R., Characterization of the techniques of pressure ejection and microiontophoresis using in vivo electrochemistry, J. Neurosci. Methods, 22 (1987) 147-159. 5 Gordon, EJ., Aortic baroreceptor reflexes are mediated by NMDA receptors in caudal ventrolateral medulla, Am. J. Physiol., 252 (1987) R628-R633. 6 Hollister, L.E., Effect of hallucinogens in humans. In B.L. Jacobs (Ed.), Hallucinogens: Neurochemical, Behavioral and Clinical Perspective, Raven, New York, 1984, pp. 19-34. 7 Honey, C.R., Miljkovic, Z. and MacDonald, J.E, Ketamine and phencyclidine cause a voltage dependent block of responses to L-aspartic acid, Neurosci. Lett., 61 (1985) 135-139. 8 Kemp, J.A., Foster, A.C., Gill, R. and Woodruff, G.N., MK801, NMDA receptors and ischemia-induced neurodegeneration, Trends Pharmacol., 8 (1987) 414-416. 9 Krebs, M.O., Kernel, M.L., Gauchy, C., Desban, M. and Glowinski, J., Glycine potentiates the NMDA-induced release of dopamine through a strychnine-insensitive site in the rat striatum, Eur. J. Pharmacol., 166 (1989) 567-570. 10 Kubo, T. and Kihara, M., Evidence of N-methyl-D-aspartate receptor-mediated modulation of the aortic baroreceptor reflex in the rat nucleus tractus solitarii, Neurosci. Lett., 87 (1988) 69-74. 11 Lin, A.M.Y., Wang, Y., Kao, J.S. and Chai, C.Y., Homocysteic acid elicits pressor responses from ventrolateral medulla and dorsomedical medulla, Brain Res. Bull., 22 (1989) 627-631. 12 Marshall, B.E. and Wollman, H., General Anesthetics. In A. Goodman-Gilman, L.S. Goodman, T.W. Rail and E Murad (Eds.), Pharmacological Basis of Therapeutics, 7th edn., Mac-

Acknowledgements. This study was supported by NSC Grant 79-0412-B016-20of Republic of China and USPHS Grant DA02429.

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