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Cardiovascular effects of NMDA in the RVLM of spontaneously hypertensive rats
BrainResearchBulletin,Vol. 37, No. 3, pp. 289-294, 1995 Copyright© 1995ElsevierScienceLtd Printedin the USA.All rightsreserved 0361-9230/95$9.50 + .00
Pergamon 0361-9230(95)00014-3
Cardiovascular Effects of NMDA in the RVLM of Spontaneously Hypertensive Rats J. C. LIN,* W. L. TSAO* AND YUN WANG1-1 Departments of *Neurology and ?Pharmacology, National Defense Medical Center, P.O. Box 90048-504, Taipei, Taiwan, 100 [Received 15 April 1994; Accepted 22 December 1994] pressure [11,12]. We, and others, previously reported that NMDA-induced cardiovascular effects can be blocked by NMDA antagonists such as 2-amino-5-phosphono-valerate (APS), MK-801, or phencyclidine (PCP) [4,22]. A similar interaction was found using electrophysiological techniques; NMDAevoked neuronal excitation in the rostral ventrolateral medulla (RVLM) was also antagonized by locally applied 2-amino-7phosphonobeptaneoate (Air/) [4], suggesting that NMDA-mediated excitation of RVLM neurons involves NMDA receptors. However, there is no direct evidence in the literature showing that hypertension induced by excitation of RVLM, such as electrical stimulation or baroreflex, is mediated through NMDA receptors in this area. Previous studies from our, and other, laboratories suggested that pressor responses induced by electrical stimulation of the sympathetic pressor area in medulla were larger in the spontaneously hypertensive rats (SHRs) than in the Wistar-Kyoto rats (WKYs) [9,13]. On the other hand, we also found that the depressor responses, which can be evoked by stimulation of the paramedian reticular nucleus, were not different between these two strains [8]. Furthermore, it has been reported that content of glutamate in the RVLM is higher in SHR compared to WKY even in the prehypertensive state [7]. It has been shown that excitatory amino acids enhance neuronal excitability by lowering the threshold for stimulation [16]. It is, thus, possible that the difference in the distribution of glutamate in the RVLM may explain the difference between SHRs and WKYs in sensitivity of the RVLM to electrical and/or chemical stimulation. Previous studies have also suggested that excitatory amino acids are involved in the baroreceptor reflex [3,6]. We found that bilateral carotid clamping-inducedhypertension was antagonized by intraventricular administration of Air/ [4]. Because carotid clamping-inducedneuronal excitation in the RVLM was also antagonized by locally applied NMDA receptor antagonists, we postulated that NMDA receptors may be involved in baroreflexinduced hypertensive responses in the RVLM. The purpose of the present study was to investigate excitatory amino acid- mediated hypertension in the RVLM of hypertensive animals. We used electrical stimulation, local NMDA application, and carotid clamping to elicit hypertensive responses. Our data suggest that NMDA may elicit a higher pressor response in the RVLM of the SHRs than in those of the WKYs.
ABSTRACT: In this study we found that cardiovascular effects were differentially regulated by N-methyl-o-aspartate (NMDA) in the rostral ventral lateral medulla (RVLM) of spontaneously hypertensive rats (SHRa) compared to their normotensive controls (Wistar-Kyoto rats, WKYs). Adult SHRs and WKYs were anesthetized with urathane, cervically vagotomized, and placed in a sterotaxic frame. We found that electrical stimulation or local application of N-mathyl-D-aspartate into the RVLM produced hypertension in both strains. Microinjection (3.5-4.0 nmol) of AP5 (2-amino-5-phosphono-valerate),an NMDA receptor antagonist, to the RVLM did not affect resting blood pressure; however, this agent antagonized hyperllmsive responses evoked by iow-threquency electrical stimulation (5-20 Hz) in both strains. The elevation in blood pressure evoked by stimulation at a higher fTequency (60 Hz) was not affected by AP5. These results suggest that NMDA receptors are involved in the low frequency, electrically evoked hypertension in both strains. We also found that SHRs had a larger pressor response to microinjection of NMDA and electrical stimulation then did WKYs. AP5 abolished the differencas in evoked hypertension between WKYs and SHRs during low-frequency (5-10 Hz) electrical stimulation. These data suggest that the hypersensitivity of RVLM to low-frequency electrical stimulation in SHRs involve NMDA receptors. We previously reported that AP7 antagonizes NMDA and carotid clamping-induced hypertension. In this study, we found that when locally applied to RVLM, AP5 antagonized hypertension evoked by clamping the carotid arteries in SHRs and WKYs. Thus, carotid clamping-induoed hypertension may also involve NMDA receptors in the RVLM. Taken together, these results suggest that NMDA receptors in the RVLM are involved in the vasomotor regulation. Furthermore, this involvement of NMDA receptors may be diffarenUally regulated among different genetic populations, because SHRs were more sensitive to the low-frequency electrical stimulation and NMDA chemical stimulation in the RVLM than WKYs. KEY WORDS: NMDA, RVLM, Hypertension, SHR.
INTRODUCTION Excitatory amino acids have been shown to regulate cardiovascular functions in medulla oblongata [2,5,10]. Topical application of N-methyl-D-aspartate (NMDA), quisqualate, and kainate to the ventral medullary surface results in an increase in blood To whom requests for reprints should be addressed. 289
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METHOD
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Animals Adult (14-20-week-old) male SHRs (n = 33) and agematched male WKYs (n = 25), weighting 2 5 0 - 3 5 0 g, were anesthetized with urethane (1.25 g/kg, IP). The femoral artery was cannulated with polyethylene catheters (PE-50, Dural Plastics and Engineering Pty. Ltd.) for measuring systemic arterial pressure (SAP), mean systemic arterial pressure (MSAP), and heart rate (HR). Systemic arterial pressure was recorded through a strain gauge transducer (Statham P23 ID). The SAP, MSAP, and HR were monitored throughout these experiments and recorded on a strip chart recorder (Gould RS 3600). The baseline systolic and mean arterial pressures in SHRs were 179 _+_ 6 mmHg and 133 _+ 3 mmHg, while those in WKYs were 123 _+ 2 and 91 _+ 2 mmHg. The animals were intubated and connected to a rodent respirator (Harvard 680, Harvard Apparatus Ltd., Edenbridge, Kent, UK) with room air (1 - 1.2 ml/100 g body weight; 6 0 - 7 0 strokes/ s). Animals were placed in a stereotaxic frame (David Kopf Instruments, Tujunga, CA). Body temperature was monitored with a thermistor probe and maintained at 37°C with a heating pad.
Electrical Stimulation Electrical stimulation was accomplished though a carbon fiber electrode (tip diameter = 100/zm) using a 10-s train of 20 #A square wave pulses at frequencies of 5, 10, 20, and 60 Hz and a duration of 0.5 ms, delivered directly to the RVLM through a stimulus isolation unit (Grass $88) and a constant-current monitor (Grass, model CCU 1A). Sterotaxic coordinates for electrode implantation in the RVLM were: 2.5 mm anterior to the obex, 2.0 mm lateral to the midline, 2.6 mm below the medullary surface (Fig. 1C), Peak hypertensive response upon electrical stimulation was analyzed against various stimulation frequencies. Relapse time between two electrical stimuli was set to 5 min. After a complete frequency-response curve was established, AP5 (3.5 nmol in 350 nl) was administered locally (see below) to the same site of RVLM, and the electrical stimulation cycle was repeated 10 min later. All the stimulated sites were marked, after each study, by electrical lesioning or local application of 0.5% fast green solution through the stimulation electrode or multibarrel pipette and were histologically verified in frozen sections.
Drug Application Drugs to be locally applied, such as N M D A (Sigma, St. Louis, MO) and AP5 (RBI, Natick, MA), were dissolved in saline (0.9% NaC1 in 10 mM phosphate buffer, pH 7.2-7.4 at 36-38°C) and applied through multibarrel pipettes to the RVLM [14]. The multibarrel micropipette and the stimulation electrode were mounted together with sticky wax (Kerr Inc., Sybron, CA); tips were separated by 50 #m. The electrode/pipette assembly was lowered into the RVLM. Local application of N M D A ( 1 0 - 2 0 0 pmol in 10-200 nl) from the multibarrel micropipettes was performed by pressure ejection using a pneumatic pump (PPM-2, Medical Systems Corp., Great Neck, NY) before and after AP5 administration (3.5 mol in 350 nl, a dose that has been shown to antagonize NMDA-mediated cardiovascular responses) [ 15,17]. The ejected volume was monitored by recording the change in the fluid meniscus in the pipette before and after ejection [23], using a dissection microscope (Olympus). The interval between two N M D A injections was set at 30 min. In some animals, AP5 was injected into R V L M bilaterally as described by different laboratories [3,17]. In brief, we implanted two micropipettes to the right and left R V L M using two sets of micromanipulator. Drugs were de-
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NMDA (pmol) FIG. 1. (A) Microejection of NMDA into the RVLM dose dependently elevates systemic arterial pressure (SAP), mean systemic arterial pressure (MSAP), and heart rate (HR). Arrows represent the time of injection of NMDA (a = 10 pmol in 10 nl; b = 25 pmol in 25 nl; c = 50 pmoi in 50 nl; d = 100 pmol in 100 nl). (B) NMDA-induced hypertensive response is dose dependent in SHR (filled circles) and WKY (open circles). Local application of NMDA into RVLM produces a greater blood pressure response in the SHRs than in the WKYs. (n = 9 for both strains; *p < 0.05, by two-way ANOVA for repeated measures and followed by Newman-Keuls post hoc analysis). In this and the following figures B.P. on the Y axis represents the difference in mean systemic arterial pressure before and after NMDA injections or electrical stimulation. Histological verification of stimulation site (filled triangle) is shown in C. Abbreviations: IO, inferior olive; MLF, medial longitudinal fasciculus; NTS, nucleus tractus solitarius; NA, nucleus ambiguus; STN, spinal trigeminal nucleus. livered from the pipettes through two separate channels of pressure microejection modules (BH-2, Medical Systems Corp., Great Neck, NY) at the same time.
Carotid Clamping The baroreceptor reflex was induced by carotid clamping as described previously [4]. In brief, the common carotid arteries were separated from the surrounding tissue bilaterally. Cervical vagotomy was performed to minimize any local mechanical stimulation. The arteries were clamped for periods of 30 s.
Statistical Analysis Results are expressed as mean _+ SEM. The data were analyzed by unpaired t-test and two-way analysis of variance (ANOVA) for repeated measures followed by the N e w m a n -
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RESULTS In 18 animals studied, we found that local application of NMDA to the RVLM increased blood pressure in a dose-dependent fashion (Fig. 1). The SHRs appeared to be more sensitive to NMDA than the WKYs. Locally applied NMDA produced a higher blood pressure in the SHRs (n = 9) than in the WKYs (n = 9). Using different stimulation frequency protocols, we found that electrically evoked hypertension was frequency dependent (Fig. 2). The SHRs were more sensitive to electrical stimulation in the RVLM, because a greater pressor response was found upon electrical stimulation in the SHRs, compared to the WKYs for a given stimulation frequency (Fig. 2). We found that the properties of hypertension induced by lowfrequency (<20 Hz) electrical stimulation in the RVLM was pharmacologically different from that induced by high-frequency (>60 Hz) electrical stimulation. Only low-frequency-evoked hypertension was antagonized by locally applied AP5 (3.5 nmol in 350 nl), at the doses that did not affect resting blood pressure, in the SHRs (n = 11) and WKYs (n = 11). High frequency (60 Hz) stimulation-mediated responses were not altered by AP5 (Figs. 3 and 4). The AP5-mediated antagonism of the low-frequency stimulation was not an artifact derived from the ejection, because saline, at the same volume, did not affect the electrically evoked responses (Figs. 3 and 4). Sixty minutes after AP5 injection, hypertensive reactions induced by either NMDA or lowfrequency electrical stimulation recovered, suggesting that the AP5-mediated antagonism was reversible (Fig. 4). AP5 not only antagonized NMDA and low-frequency electrical stimulation-inducedpressor responses, but it also reduced the difference in the hypertension between WKYs and SHRs evoked by low-frequency (5-10 Hz) electrical stimulation (Fig. 5). This result suggests that the supersensitivityobserved for low-
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STIMULATION FREQUENCY (Hz) FIG. 3. Hypertensioninduced by low-frequencyelectrical stimulationat RVLM is antagonized by locally applied AP5 in both the SHRs and WKYs. Local application of AP5 (3.5 nmol in 350 nl) to the RVLM antagonizes hypertension induced by low-frequency electrical stimulation (5-20 Hz) in the SHRs (A, n = 11) and WKYs (B, n = 11). Saline (n = 3), injection in the same volume, does not antagonize either highor low-frequency stimulation-evokedhypertension. (*p < 0.05, **p < 0.01, by two-way ANOVA for repeated measures and followed by Newman-Keuls post hoc analysis). frequency electrical stimulation in SHR involved NMDA receptors. We previously reported that intraventricularadministration of NMDA antagonists diminished carotid clamping-inducedhypertension in Sprague-Dawley rats. In the present study, we found that carotid clamping induced a similar hypertensive response in both SHRs (n = 13) and WKYs (n = 5; Fig. 6B). Bilateral injection of AP5 (3.5-4.0 nmol in 350-400 nl, each side) into the RVLM antagonized clamping-induced hypertension in WKYs and SHRs. The clamping-induced hypertensive responses gradually recovered over 30 rain after AP5 injection (Fig. 6A). We also found that carotid clamping-induced hypertension was antagonized by locally applied AP5 in the WKY and SHR to the same extent.
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FIG. 2. SHR produces a higher pressure response upon electrical stimulation than does WKY. Stimulation(20/~A, 5 ms, square wave) is given directly to the RVLM at different frequencies in one WKY rat (A-D) and one SHR (E-H). Note the scales of A-D are different from that of E-H. Horizontal bars under each tracing represent the time of stimulation. (Stimulation frequency: A, E = 5 Hz; B, F = 10 Hz; C, G = 20 Hz; D, H = 60 Hz.)
DISCUSSION In the present study, we found that local application of NMDA to the RVLM produced hypertensive responses in SHRs and WKYs and that SHRs were more sensitive to this drug than WKYs. Similar hypertensive responses were also found in SHRs following injection of other excitatory amino acids, such as glu-
292
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and WKYs. The effects of low-frequency electrical stimulation was antagonized by locally applied AP5. This suggests that lowfrequency electrical stimulation may induce release of excitatory amino acids that selectively activate N M D A receptors and result in hypertension. On the other hand, high-frequency stimulationmediated responses were not sensitive to AP5 in either strain. It is possible that high-frequency electrical stimulation may activate other pathways nearby the RVLM. It has been reported that the threshold current required to evoke blood pressure responses in the subretrofacial nucleus of RVLM was similar between WKY and SHR [13]. We found that, using differential stimulation frequency protocols, SHRs produce greater pressor responses. Taken together, these data suggest that the RVLM neurons of SHRs may be more sensitive to changes of frequency, but not current, during stimulation. Although hypertension induced by locally applied N M D A or electrical stimulation was greater in the SHRs, the regulation of these two responses may not be the same. Differences in responses between the WKYs and SHRs were found at high doses
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FIG. 4. NMDA and low-frequency stimulation-evoked hypertensive responses are antagonized by locally applied AP5. A - D represents the pressor response obtained from four different animals. Local application of NMDA (200 pmol in 200 nl) to the RVLM produces an elevation of mean arterial pressure (AI, control) and systemic arterial pressure (A2, control). Pretreatment with AP5 (3.5 nmol in 350 nl; 10 min prior to the NMDA ejection), antagonizes NMDA-induced hypertension (AI, A2, and AP5). Pretreatment with saline does not affect this NMDA-induced pressor response (A I and A2: saline vs. control). NMDA-evoked pressor response recovered 60 min after AP5 application (A 1 and A2, recovered). (B-D) Electrical stimulation of the RVLM (stimulation frequency: B = 10 Hz; C = 20 Hz; D = 60 Hz) also produces hypertension. Pretreatment with AP5 antagonizes the pressor responses induced by low-frequency electrical stimulation (B, C. AP5). High-frequency-evoked pressor response is not antagonized by AP5 (D, AP5).
tamate, kainate, and quisqualate, into the RVLM [13]. These findings suggest that SHRs and WKYs have a generalized differential sensitivity to excitatory amino acids in the RVLM. It has been known that the RVLM and caudal ventral lateral medulla (CVLM) contain synapses of excitatory amino acids [3,17-19]. However, the cardiovascular effects of excitatory amino acids in RVLM and CVLM are different. We and others found that injection of N M D A into RVLM [2,3,5,9,13,17-19] or CVLM [3,5,17]-induced hypertensive or hypotensive responses, respectively. Bilateral injection of AP5, an N M D A receptor antagonist, did not lower the basal blood pressure in either SHR or WKY. Similar results have been reported using local application of kynurenic acid in the RVLM [ 18]. These data suggest that N M D A receptors in RVLM are not tonically active. In contrast, bilateral injection of N M D A antagonists to the CVLMinduced hypertensive responses [3,5,17]. Because blocking N M D A receptors in the CVLM, not in the RVLM, elicits cardiovascular response, it is possible that N M D A receptors of the CVLM, unlike those in the RVLM, are tonically active. It has been shown that the RVLM receives excitatory amino acid inputs from several brain areas, such as hypothalamus or nucleus tractus solitarii [17,18]. SHRs show more glutamate immunoreactivity in the RVLM than do WKYs [19]. Stimulation of RVLM may induce release of excitatory amino acids in this area [20]. In the present study, we found that electrical stimulation of the RVLM produced hypertensive reactions in both SHRs
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STIMULATION FREQUENCY (Hz) FIG. 5. Increased sensitivity to electrical stimulation in SHRs involves NMDA receptors. (A) Before AP5 injection, SHRs (n = 11) are more sensitive than WKYs (n = 11) to electrical stimulation (*p < 0.05; **p < 0.01, by two-way ANOVA for repeated measures and followed by Newman-Keuls post hoc method). (B) After local RVLM application of AP5, the difference in evoked hypertension between WKYs and SHRs is abolished at low-frequency (5-10 Hz) electrical stimulation. Highfrequency-evoked responses, however, are not affected by AP5.
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FIG. 6. AP5 in RVLM antagonizescarotid clamping-inducedhypertension in both SHR and WKY. (A) Bilateral, local application of saline 400 ni to the RVLM does not alter carotid clamping-mediatedelevation of systemic and mean arterial pressure (b vs. a). However, bilateral injection of AP5 (4.0 nmol in 400 nl, bilateral) into the RVLM antagonizes clamping-inducedhypertension (c: 10 rain d: 20 min; e: 30 min after AP5 injection).Horizontalbars under each tracing represent the duration of carotid clamping. (B) Bar graphs of responses to carotid clamping in SHRs (n = 13) and WKYs (n = 5). B.P. represents the difference of basal mean blood pressure and maximalpressure response during carotid clamping. Local applicationof AP5 antagonizesclamping-mediatedhypertension in WKYs and SHRs to the same extent. (**p < 0.01, by unpaired t-test). of NMDA and low levels of electrical stimulation, whereas low doses of NMDA or high levels of electrical stimulation did not elicit differences between the WKYs and the SHRs. It has also been reported that the spontaneous release of aspartate from the presynaptic nerve terminals is less in the RVLM of SHRs as compared to that of WKYs [20], suggesting that differences in presynaptic excitatory amino acid regulatory mechanisms exist in WKYs vs. SHRs. It is, thus, possible that local application of NMDA and electrical stimulation may involve different pre- and postsynaptic mechanisms in the RVLM in these two strains. We found that low-frequency electrical stimulation-evoked hypertensive responses in SHRs and WKYs were abolished after AP5 application. This suggests that the higher sensitivity to the low-frequency electrical stimulation in SHRs is mediated through NMDA receptors. In the present study, we used a low concentration (1 mM NMDA, 10 mM AP5) but relatively high volume ( 1 0 - 2 0 0 nl
NMDA, 350-400 nl AP5) of drugs for local application. Similar experiments were used in other laboratories [1,2,8,15]. The use of high volume is necessary to compensate the osmolaric effects of drugs, because a very concentrated solution (up to 1 M) was required for low volume application [2]. In the control experiment, we also found that application of 350 nl of saline to the RVLM did not alter chemical or electrically evoked pressor responses, and furthermore, local application of 350 nl of fast green solution to the RVLM did not result in spreading the dye to the adjacent brain areas, suggesting that actions of drugs used in the present study have effects limited to the RVLM. Similar control experiments have been demonstrated in other laboratories. With 200 nl of 0.5% fast green injected locally to the RVLM, a stained spot was produced in the brain tissue 0.53 ___0.05 mm in diameter [2]. Our previous findings indicated that the alternation of blood pressure induced by carotid clamping for less than 100 s is little influenced by decerebration and is abolished by denervation of bilateral glossopharyngeal nerves in vagotomized animals [4]. These findings suggest that the changes of blood pressure elicited by clamping for less than 100 s in the vagotomized animals is primarily mediated through the baroreceptor reflex. This carotid clamping-inducedhypertension was antagonized by intraventricular injection of AP7 or PCP [4]. In the present study, we found that after local application of AP5 to the RVLM, the clampinginduced hypertension was antagonized to a same extent in WKYs and SHRs. This suggests that carotid clamping-induced hypertension involves NMDA receptors in the RVLM. However, we also found that SHRs did not show differential sensitivity to this NMDA receptor-mediated pressor response in the carotid clamping experiment, which is different from the NMDA-mediated hypertension reactions seen during local NMDA application or electrical stimulation. It has been reported that NMDA-mediated responses are modulated by additional chemical species, such as Mg 2+, and glycine [21]. Baroreceptor reflexes, on the other hand, may activate other neurotransmitter pathways or changes in the extracellular environment. It is possible that the lack of differential sensitivity of the NMDA receptor-mediated reactions during carotid clamping between SHRs and WKYs may derive from non-NMDA modulatory mechanisms, which may also be differentially regulated in WKYs and SHRs. The lack of greater NMDA receptor-dependent increase in blood pressure with carotid occlusion in SHRs suggests that the baroreflex is equally effective in the hypertensive or normotensive animals or patients. Our data indicates that low-frequency (5-20 Hz) electrical stimulation of the RVLM, which increases blood pressure 40 mmHg in WKYs and 60 mmHg in SHRs, involves NMDA. On the other hand, high-frequency (>60 Hz) electrical stimulation, which increases blood pressure 80 mmHg in WKYs and 95 mmHg in SHRs, is less affected by NMDA receptor antagonists. Because physiological stimulation seldom induces pressor response higher than 80-95 mmHg, our data suggest that the NMDA-mediated effect of low-frequency electrical stimulation is physiologically relevant. In conclusion, the present study indicated that NMDA receptors in the RVLM are involved in blood pressure regulation. SHR, compared to WKY, has a higher sensitivity to the lowfrequency electrical stimulation and NMDA chemical stimulation in the RVLM. It is possible that the sensitivity of NMDA receptor-mediated pressor response in the RVLM may contribute to the hypertension observed in SHR. ACKNOWLEDGEMENT
This work was supported by Grant NSC 83-0420-B016-35 of the Republic of China.
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13. Miura, M.; Takayama, K.; Okada, J. Difference in sensitivity of cardiovascular and respiratory control neurons in the subretrofacial nucleus to glutamate receptor subtype agonists in SHR, WKY and cats. J. Auton. Nerv. Syst. 36:1-12; 1991. 14. Palmer, M. R.; Wuerthele, S. M.; Hoffer, B. J. Physical and physiological characteristics of micropressure ejection of drugs from multibarreled pipettes. Neuropharmacology 19:931-938; 1980. 15. Ross, C. A.; Ruggiero, D. A.; Joh, T. H.; Park, D. H.; Reis, D. J. Adrenaline synthesizing neurons in the rostral ventrolateral medulla: A possible role in tonic vasomotor control. Brain Res. 273:356-361 ; 1983. 16. Soltesz, I.; Lightowler, S.; Leresche, N.; Jassik-Gerschenfeld, D.; Pollard, C. E.; Crunelli, V. Two inward currents and the transformation of low-frequency oscillations of rat and cat thalamocortical cells. J. Physiol. (Lond.)441:175-197; 1991. 17. Somogyi, P.; Minson, J. B.; Morilak, D.; Llewellyn-Smith, 1.; MclIhinney, J. R. A.; Chalmers, J. Evidence for an excitatory amino acid pathway in the brain stem and for its involvement in cardiovascular control. Brain Res. 496:401-407; 1989. 18. Sun, M. K.; Guyenet, P. G. Hypotbalamic glutamatergic input to medullary sympathoexcitatory neurons in rats. Am. J. Physiol. 251 :R798-R810; 1986. 19. Takayama, K.; Miura, M. Difference in distribution of glutamateimmunoreactive neurons projecting into the subretrofacial nucleus in the rostral ventrolateral medulla of SHR and WKY: A doublelabeling study. Brain Res. 570:259-266; 1992. 20. Tingley, F. D.; Arueric, S. P. Evidence for clonidine presynaptically modulating amino acid release in the rostral ventral medulla: Role in hypertension. Brain. Res. 537:175-181; 1990. 21. Varuer, K. J.; Rutherford, D. S.; Vasquez, E. C.; Brody, M. J. Identification of cardiovascular neurons in the rostral ventromedial medulla in anesthetized rats. Hypertension 19:II193-I1197; 1992. 22. Verberne, A. J. M.; Costa, M.; Lewis, S. J.; Louis, W. J.; Beart, P. M. The N-methyl-D-aspartate (NMDA) receptor antagonist MK801, attenuates the Bezold-Jarisch reflex in the anesthetized rat. Neuropharmacology 26:1243-1246; 1987. 23. Wang, Y.; Wang, S. D.; Lin, S. Z.; Liu, J. C. Restoration of dopamine overflow and clearance from 6-OHDA lesion rat striatum reinnervated by fetal mesencephalic graft. J. Pharmacol. Exp. Ther. 270:814-821; 1994.
Pergamon 0361-9230(95)00014-3
Cardiovascular Effects of NMDA in the RVLM of Spontaneously Hypertensive Rats J. C. LIN,* W. L. TSAO* AND YUN WANG1-1 Departments of *Neurology and ?Pharmacology, National Defense Medical Center, P.O. Box 90048-504, Taipei, Taiwan, 100 [Received 15 April 1994; Accepted 22 December 1994] pressure [11,12]. We, and others, previously reported that NMDA-induced cardiovascular effects can be blocked by NMDA antagonists such as 2-amino-5-phosphono-valerate (APS), MK-801, or phencyclidine (PCP) [4,22]. A similar interaction was found using electrophysiological techniques; NMDAevoked neuronal excitation in the rostral ventrolateral medulla (RVLM) was also antagonized by locally applied 2-amino-7phosphonobeptaneoate (Air/) [4], suggesting that NMDA-mediated excitation of RVLM neurons involves NMDA receptors. However, there is no direct evidence in the literature showing that hypertension induced by excitation of RVLM, such as electrical stimulation or baroreflex, is mediated through NMDA receptors in this area. Previous studies from our, and other, laboratories suggested that pressor responses induced by electrical stimulation of the sympathetic pressor area in medulla were larger in the spontaneously hypertensive rats (SHRs) than in the Wistar-Kyoto rats (WKYs) [9,13]. On the other hand, we also found that the depressor responses, which can be evoked by stimulation of the paramedian reticular nucleus, were not different between these two strains [8]. Furthermore, it has been reported that content of glutamate in the RVLM is higher in SHR compared to WKY even in the prehypertensive state [7]. It has been shown that excitatory amino acids enhance neuronal excitability by lowering the threshold for stimulation [16]. It is, thus, possible that the difference in the distribution of glutamate in the RVLM may explain the difference between SHRs and WKYs in sensitivity of the RVLM to electrical and/or chemical stimulation. Previous studies have also suggested that excitatory amino acids are involved in the baroreceptor reflex [3,6]. We found that bilateral carotid clamping-inducedhypertension was antagonized by intraventricular administration of Air/ [4]. Because carotid clamping-inducedneuronal excitation in the RVLM was also antagonized by locally applied NMDA receptor antagonists, we postulated that NMDA receptors may be involved in baroreflexinduced hypertensive responses in the RVLM. The purpose of the present study was to investigate excitatory amino acid- mediated hypertension in the RVLM of hypertensive animals. We used electrical stimulation, local NMDA application, and carotid clamping to elicit hypertensive responses. Our data suggest that NMDA may elicit a higher pressor response in the RVLM of the SHRs than in those of the WKYs.
ABSTRACT: In this study we found that cardiovascular effects were differentially regulated by N-methyl-o-aspartate (NMDA) in the rostral ventral lateral medulla (RVLM) of spontaneously hypertensive rats (SHRa) compared to their normotensive controls (Wistar-Kyoto rats, WKYs). Adult SHRs and WKYs were anesthetized with urathane, cervically vagotomized, and placed in a sterotaxic frame. We found that electrical stimulation or local application of N-mathyl-D-aspartate into the RVLM produced hypertension in both strains. Microinjection (3.5-4.0 nmol) of AP5 (2-amino-5-phosphono-valerate),an NMDA receptor antagonist, to the RVLM did not affect resting blood pressure; however, this agent antagonized hyperllmsive responses evoked by iow-threquency electrical stimulation (5-20 Hz) in both strains. The elevation in blood pressure evoked by stimulation at a higher fTequency (60 Hz) was not affected by AP5. These results suggest that NMDA receptors are involved in the low frequency, electrically evoked hypertension in both strains. We also found that SHRs had a larger pressor response to microinjection of NMDA and electrical stimulation then did WKYs. AP5 abolished the differencas in evoked hypertension between WKYs and SHRs during low-frequency (5-10 Hz) electrical stimulation. These data suggest that the hypersensitivity of RVLM to low-frequency electrical stimulation in SHRs involve NMDA receptors. We previously reported that AP7 antagonizes NMDA and carotid clamping-induced hypertension. In this study, we found that when locally applied to RVLM, AP5 antagonized hypertension evoked by clamping the carotid arteries in SHRs and WKYs. Thus, carotid clamping-induoed hypertension may also involve NMDA receptors in the RVLM. Taken together, these results suggest that NMDA receptors in the RVLM are involved in the vasomotor regulation. Furthermore, this involvement of NMDA receptors may be diffarenUally regulated among different genetic populations, because SHRs were more sensitive to the low-frequency electrical stimulation and NMDA chemical stimulation in the RVLM than WKYs. KEY WORDS: NMDA, RVLM, Hypertension, SHR.
INTRODUCTION Excitatory amino acids have been shown to regulate cardiovascular functions in medulla oblongata [2,5,10]. Topical application of N-methyl-D-aspartate (NMDA), quisqualate, and kainate to the ventral medullary surface results in an increase in blood To whom requests for reprints should be addressed. 289
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LIN, T S A O AND WANG
METHOD
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Animals Adult (14-20-week-old) male SHRs (n = 33) and agematched male WKYs (n = 25), weighting 2 5 0 - 3 5 0 g, were anesthetized with urethane (1.25 g/kg, IP). The femoral artery was cannulated with polyethylene catheters (PE-50, Dural Plastics and Engineering Pty. Ltd.) for measuring systemic arterial pressure (SAP), mean systemic arterial pressure (MSAP), and heart rate (HR). Systemic arterial pressure was recorded through a strain gauge transducer (Statham P23 ID). The SAP, MSAP, and HR were monitored throughout these experiments and recorded on a strip chart recorder (Gould RS 3600). The baseline systolic and mean arterial pressures in SHRs were 179 _+_ 6 mmHg and 133 _+ 3 mmHg, while those in WKYs were 123 _+ 2 and 91 _+ 2 mmHg. The animals were intubated and connected to a rodent respirator (Harvard 680, Harvard Apparatus Ltd., Edenbridge, Kent, UK) with room air (1 - 1.2 ml/100 g body weight; 6 0 - 7 0 strokes/ s). Animals were placed in a stereotaxic frame (David Kopf Instruments, Tujunga, CA). Body temperature was monitored with a thermistor probe and maintained at 37°C with a heating pad.
Electrical Stimulation Electrical stimulation was accomplished though a carbon fiber electrode (tip diameter = 100/zm) using a 10-s train of 20 #A square wave pulses at frequencies of 5, 10, 20, and 60 Hz and a duration of 0.5 ms, delivered directly to the RVLM through a stimulus isolation unit (Grass $88) and a constant-current monitor (Grass, model CCU 1A). Sterotaxic coordinates for electrode implantation in the RVLM were: 2.5 mm anterior to the obex, 2.0 mm lateral to the midline, 2.6 mm below the medullary surface (Fig. 1C), Peak hypertensive response upon electrical stimulation was analyzed against various stimulation frequencies. Relapse time between two electrical stimuli was set to 5 min. After a complete frequency-response curve was established, AP5 (3.5 nmol in 350 nl) was administered locally (see below) to the same site of RVLM, and the electrical stimulation cycle was repeated 10 min later. All the stimulated sites were marked, after each study, by electrical lesioning or local application of 0.5% fast green solution through the stimulation electrode or multibarrel pipette and were histologically verified in frozen sections.
Drug Application Drugs to be locally applied, such as N M D A (Sigma, St. Louis, MO) and AP5 (RBI, Natick, MA), were dissolved in saline (0.9% NaC1 in 10 mM phosphate buffer, pH 7.2-7.4 at 36-38°C) and applied through multibarrel pipettes to the RVLM [14]. The multibarrel micropipette and the stimulation electrode were mounted together with sticky wax (Kerr Inc., Sybron, CA); tips were separated by 50 #m. The electrode/pipette assembly was lowered into the RVLM. Local application of N M D A ( 1 0 - 2 0 0 pmol in 10-200 nl) from the multibarrel micropipettes was performed by pressure ejection using a pneumatic pump (PPM-2, Medical Systems Corp., Great Neck, NY) before and after AP5 administration (3.5 mol in 350 nl, a dose that has been shown to antagonize NMDA-mediated cardiovascular responses) [ 15,17]. The ejected volume was monitored by recording the change in the fluid meniscus in the pipette before and after ejection [23], using a dissection microscope (Olympus). The interval between two N M D A injections was set at 30 min. In some animals, AP5 was injected into R V L M bilaterally as described by different laboratories [3,17]. In brief, we implanted two micropipettes to the right and left R V L M using two sets of micromanipulator. Drugs were de-
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Carotid Clamping The baroreceptor reflex was induced by carotid clamping as described previously [4]. In brief, the common carotid arteries were separated from the surrounding tissue bilaterally. Cervical vagotomy was performed to minimize any local mechanical stimulation. The arteries were clamped for periods of 30 s.
Statistical Analysis Results are expressed as mean _+ SEM. The data were analyzed by unpaired t-test and two-way analysis of variance (ANOVA) for repeated measures followed by the N e w m a n -
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RESULTS In 18 animals studied, we found that local application of NMDA to the RVLM increased blood pressure in a dose-dependent fashion (Fig. 1). The SHRs appeared to be more sensitive to NMDA than the WKYs. Locally applied NMDA produced a higher blood pressure in the SHRs (n = 9) than in the WKYs (n = 9). Using different stimulation frequency protocols, we found that electrically evoked hypertension was frequency dependent (Fig. 2). The SHRs were more sensitive to electrical stimulation in the RVLM, because a greater pressor response was found upon electrical stimulation in the SHRs, compared to the WKYs for a given stimulation frequency (Fig. 2). We found that the properties of hypertension induced by lowfrequency (<20 Hz) electrical stimulation in the RVLM was pharmacologically different from that induced by high-frequency (>60 Hz) electrical stimulation. Only low-frequency-evoked hypertension was antagonized by locally applied AP5 (3.5 nmol in 350 nl), at the doses that did not affect resting blood pressure, in the SHRs (n = 11) and WKYs (n = 11). High frequency (60 Hz) stimulation-mediated responses were not altered by AP5 (Figs. 3 and 4). The AP5-mediated antagonism of the low-frequency stimulation was not an artifact derived from the ejection, because saline, at the same volume, did not affect the electrically evoked responses (Figs. 3 and 4). Sixty minutes after AP5 injection, hypertensive reactions induced by either NMDA or lowfrequency electrical stimulation recovered, suggesting that the AP5-mediated antagonism was reversible (Fig. 4). AP5 not only antagonized NMDA and low-frequency electrical stimulation-inducedpressor responses, but it also reduced the difference in the hypertension between WKYs and SHRs evoked by low-frequency (5-10 Hz) electrical stimulation (Fig. 5). This result suggests that the supersensitivityobserved for low-
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DISCUSSION In the present study, we found that local application of NMDA to the RVLM produced hypertensive responses in SHRs and WKYs and that SHRs were more sensitive to this drug than WKYs. Similar hypertensive responses were also found in SHRs following injection of other excitatory amino acids, such as glu-
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tamate, kainate, and quisqualate, into the RVLM [13]. These findings suggest that SHRs and WKYs have a generalized differential sensitivity to excitatory amino acids in the RVLM. It has been known that the RVLM and caudal ventral lateral medulla (CVLM) contain synapses of excitatory amino acids [3,17-19]. However, the cardiovascular effects of excitatory amino acids in RVLM and CVLM are different. We and others found that injection of N M D A into RVLM [2,3,5,9,13,17-19] or CVLM [3,5,17]-induced hypertensive or hypotensive responses, respectively. Bilateral injection of AP5, an N M D A receptor antagonist, did not lower the basal blood pressure in either SHR or WKY. Similar results have been reported using local application of kynurenic acid in the RVLM [ 18]. These data suggest that N M D A receptors in RVLM are not tonically active. In contrast, bilateral injection of N M D A antagonists to the CVLMinduced hypertensive responses [3,5,17]. Because blocking N M D A receptors in the CVLM, not in the RVLM, elicits cardiovascular response, it is possible that N M D A receptors of the CVLM, unlike those in the RVLM, are tonically active. It has been shown that the RVLM receives excitatory amino acid inputs from several brain areas, such as hypothalamus or nucleus tractus solitarii [17,18]. SHRs show more glutamate immunoreactivity in the RVLM than do WKYs [19]. Stimulation of RVLM may induce release of excitatory amino acids in this area [20]. In the present study, we found that electrical stimulation of the RVLM produced hypertensive reactions in both SHRs
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NMDA, 350-400 nl AP5) of drugs for local application. Similar experiments were used in other laboratories [1,2,8,15]. The use of high volume is necessary to compensate the osmolaric effects of drugs, because a very concentrated solution (up to 1 M) was required for low volume application [2]. In the control experiment, we also found that application of 350 nl of saline to the RVLM did not alter chemical or electrically evoked pressor responses, and furthermore, local application of 350 nl of fast green solution to the RVLM did not result in spreading the dye to the adjacent brain areas, suggesting that actions of drugs used in the present study have effects limited to the RVLM. Similar control experiments have been demonstrated in other laboratories. With 200 nl of 0.5% fast green injected locally to the RVLM, a stained spot was produced in the brain tissue 0.53 ___0.05 mm in diameter [2]. Our previous findings indicated that the alternation of blood pressure induced by carotid clamping for less than 100 s is little influenced by decerebration and is abolished by denervation of bilateral glossopharyngeal nerves in vagotomized animals [4]. These findings suggest that the changes of blood pressure elicited by clamping for less than 100 s in the vagotomized animals is primarily mediated through the baroreceptor reflex. This carotid clamping-inducedhypertension was antagonized by intraventricular injection of AP7 or PCP [4]. In the present study, we found that after local application of AP5 to the RVLM, the clampinginduced hypertension was antagonized to a same extent in WKYs and SHRs. This suggests that carotid clamping-induced hypertension involves NMDA receptors in the RVLM. However, we also found that SHRs did not show differential sensitivity to this NMDA receptor-mediated pressor response in the carotid clamping experiment, which is different from the NMDA-mediated hypertension reactions seen during local NMDA application or electrical stimulation. It has been reported that NMDA-mediated responses are modulated by additional chemical species, such as Mg 2+, and glycine [21]. Baroreceptor reflexes, on the other hand, may activate other neurotransmitter pathways or changes in the extracellular environment. It is possible that the lack of differential sensitivity of the NMDA receptor-mediated reactions during carotid clamping between SHRs and WKYs may derive from non-NMDA modulatory mechanisms, which may also be differentially regulated in WKYs and SHRs. The lack of greater NMDA receptor-dependent increase in blood pressure with carotid occlusion in SHRs suggests that the baroreflex is equally effective in the hypertensive or normotensive animals or patients. Our data indicates that low-frequency (5-20 Hz) electrical stimulation of the RVLM, which increases blood pressure 40 mmHg in WKYs and 60 mmHg in SHRs, involves NMDA. On the other hand, high-frequency (>60 Hz) electrical stimulation, which increases blood pressure 80 mmHg in WKYs and 95 mmHg in SHRs, is less affected by NMDA receptor antagonists. Because physiological stimulation seldom induces pressor response higher than 80-95 mmHg, our data suggest that the NMDA-mediated effect of low-frequency electrical stimulation is physiologically relevant. In conclusion, the present study indicated that NMDA receptors in the RVLM are involved in blood pressure regulation. SHR, compared to WKY, has a higher sensitivity to the lowfrequency electrical stimulation and NMDA chemical stimulation in the RVLM. It is possible that the sensitivity of NMDA receptor-mediated pressor response in the RVLM may contribute to the hypertension observed in SHR. ACKNOWLEDGEMENT
This work was supported by Grant NSC 83-0420-B016-35 of the Republic of China.
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