Role of the Rostral Ventrolateral Medulla in Maintenance of Blood Pressure in Rats With Goldblatt Hypertension
Abstract The aim of the present study was to examine the participation of the rostral ventrolateral medulla (RVLM) in the maintenance of hypertension in rats submitted to the renovascular Goldblatt (two-kidney, one clip) procedure. We inhibited or stimulated this area with the use of drugs such as glycine, l-glutamate, or kynurenic acid. (1) Bilateral microinjection of glycine (100 nmol, 200 nL, n=13) into the RVLM of hypertensive rats produced a decrease in mean arterial blood pressure (MAP) from 177.2±29.3 to 102.3±20.9 mm Hg (P<.05), which was similar to the decrease produced by intravenous administration of hexamethonium. The inhibition of RVLM with glycine in normotensive rats produced a decrease in MAP from 106±17.1 to 59.7±7.3 mm Hg (P<.05, n=9). (2) An impressive increase in MAP from 153.3±16.3 to 228±34.9 mm Hg (P<.05) occurred in hypertensive rats after microinjection of l-glutamate (50 nmol, 200 nL, n=6) into the RVLM. The same procedure caused a significant but less intense increase in MAP from 105±13.8 to 148.3±24.9 mm Hg in normotensive rats (P<.05, n=6). (3) A decrease in MAP from 151.6±25.3 to 96.8±22.5 mm Hg occurred in hypertensive rats after microinjection of the broad-spectrum glutamate antagonist kynurenic acid (4 nmol, 200 nL, n=6) into the RVLM, whereas the same procedure did not change MAP in normotensive animals (n=6). Heart rate was not significantly affected in any group. Together these results show that the activity of RVLM neurons is important in the maintenance of arterial blood pressure in Goldblatt hypertensive rats and probably indicate a change in the sensitivity and/or number of glutamatergic receptors in this area after the development of hypertension.
To elucidate the mechanisms involved in the development of hypertension, the Goldblatt model has been one of the most frequently studied models in medical science. Although there is still controversy concerning the role of the central nervous system in the production and maintenance of hypertension, evidence accumulated during the past 20 years supports the concept that neurogenic mechanisms participate in renal hypertension.1 Moreover, there is experimental evidence that the central pressor actions of angiotensin II probably contribute to several forms of experimental hypertension.2 Lesions of the anteroventral portion of the third ventricle protect against the development of or reverse hypertension once it is established in different forms of experimental models.3 4
It has also been established that neurons located in the ventrolateral medulla are important in the maintenance and reflex control of ABP and also in sympathetic activity.5 6 The RVLM is a well-known pressor area that contains neurons that project directly to the sympathetic preganglionic neurons.7 However, the role of this area in ABP maintenance in animals with renovascular hypertension has not been fully explored. The aim of the present study was to contribute to the understanding of the role of RVLM activity in rats with high blood pressure due to the 2K1C Goldblatt procedure.
The study was performed on 46 male Wistar rats weighing 150 to 200 g from the Central Animal House of the Universidade Federal de São Paolo–Escola Paulista de Medicina. The left renal artery was partially obstructed with a silver clip to obtain 2K1C Goldblatt renovascular hypertension. After 6 weeks, rats were anesthetized with sodium pentobarbital 40 mg/kg, and cannulas were implanted into the femoral vein and artery and exteriorized dorsally. Twenty-four hours after the above procedure was completed, ABP and heart rate were recorded in awake, free-moving rats, and then the animals were anesthetized with urethane 1.2 g/kg IV, divided into 3 doses. The trachea was cannulated for artificial ventilation, and pulsatile ABP was recorded by using a P23XL transducer (Statham Instruments Division, Gould Inc) connected to an RS3400 recorder (Record System Division, Gould Inc). MAP was obtained by filtering the ABP signal and heart rate with a cardiotachometer (ECG/Biotach, Gould Inc) triggered by the pulse wave. Body temperature was maintained at 37°C with the use of a heating table.
Rats were placed prone in a stereotaxic apparatus (David Kopf Instruments) with the bite bar 12 mm below the interaural line. An occipital craniotomy was performed to expose the dorsal surface of the brain stem and cerebellum. The dura was opened and retracted exposing the obex, whose vertex was taken as a landmark for the stereotaxic coordinates.
Drugs were microinjected bilaterally into the RVLM through micropipettes. Guide cannulas were directed to the desired stereotaxic position (anteroposterior, 3.0 mm rostral and 1.8 mm lateral to the obex). Vertical positioning was obtained by slowly lowering both the micropipette and the guide cannula until a slight displacement between them was observed. Micropipettes were connected to Hamilton microsyringes. Microinjections consisted of 200 nL saline in which glycine 100 nmol, l-glutamate 50 nmol, or kynurenic acid 2 nmol was dissolved. The pH of all solutions was adjusted to 7.4. Three injections at most were made at the same site in each experiment. Microinjections of vehicle alone (200 nL) produced no change in ABP or heart rate in hypertensive or normotensive rats. At the end of the experiments, 200 nL 2% Evans blue dye was injected into the RVLM. Rats were euthanatized with an overdose of urethane, and injection sites were evaluated by dye diffusion into the ventral surface and plotted on schematic diagrams as previously described.8 Fig 1⇓ is representative of the dye distribution.
Changes induced by microinjection of drugs into the RVLM were analyzed by Student’s paired t test. Data are reported as mean±SD. A value of P<.05 was considered significant.
Effects of Bilateral Microinjection of Glycine Into the RVLM
In rats with 2K1C hypertension, bilateral application of glycine (100 nmol, 200 nL, n=13) into the RVLM produced a depressor response in MAP (basal 177.2±29.3 mm Hg, response 102.3±20.9 mm Hg, P<.05) as shown in Fig 2A⇓. The onset was 45.8±35.8 seconds after application; the response peaked at 3.3±1.7 minutes and remained below the resting level for 32.6±16.8 minutes. Bradycardia also was observed but was not significant compared with the basal heart rate (basal 399.6±58.7 beats per minute, response 340.8±50.1 beats per minute). In normotensive sham rats the same application decreased blood pressure (control 106.2±17.1 mm Hg, response 59.7±7.3 mm Hg, P<.05, n=9) as shown in Fig 2C⇓, accompanied by bradycardia (control 438.9±19.5 beats per minute, response 377.2±55.5 beats per minute, P>.05). The onset was 15.6±14.0 seconds after application; the response peaked at 2.2±0.7 minutes and remained below the control level for 10.6±4.8 minutes. For comparison, a ganglion blocker, hexamethonium (40 mg/kg IV), was injected into both groups. The same response obtained with glycine microinjection was observed, as shown in Fig 2B⇓ and 2D⇓. In 2K1C rats after hexamethonium a further blood pressure decrease was obtained with captopril 30 mg/kg IV, resulting in ABP of the same level as in normotensive rats treated with hexamethonium.
Effects of Bilateral Microinjection of l-Glutamate Into the RVLM
In 2K1C rats injection of l-glutamate (50 nmol, 200 nL) into the RVLM provoked a significant increase in MAP (basal 153.3±16.3 mm Hg, response 228.0±34.9 mm Hg, P<.05, n=6) as shown in Fig 3A⇓ and a small, nonsignificant decrease in heart rate (basal 414.3±55.5 beats per minute, response 405.0±48.1 beats per minute). The response started during the microinjection period, peaked at 31.7±14.7 seconds, and remained above the basal level for 2.8±1.6 minutes. When glutamate was microinjected into normal rats it produced a significant but less intense increase in MAP (control 105.0±13.8 mm Hg, response 148.3±24.9 mm Hg, P<.05, n=6) as shown in Fig 3C⇓. Heart rate was slightly decreased (control 380.0±49.0 beats per minute, response 363.3±63.5 beats per minute). The response started at 9.0±13.4 seconds, peaked at 1.5±1.0 minutes, and remained above the basal level for 5.3±2.4 minutes.
Effects of Bilateral Microinjection of the Broad-Spectrum Glutamate Antagonist Kynurenic Acid Into the RVLM
Bilateral microinjection of kynurenic acid (2 nmol, 200 nL), a broad-spectrum glutamate antagonist, into the RVLM of hypertensive rats induced a significant decrease in MAP (basal 151.6±25.3 mm Hg, response 96.8±22.5 mm Hg, P<.05, n=6) as shown in Fig 3B⇑ and a nonsignificant decrease in heart rate (basal 371±78.7 beats per minute, response 337±78.9 beats per minute). The response started at 1.8±0.9 minutes and peaked at 5.7±2.1 minutes, and a partial recovery occurred after 28.7±16.6 minutes. When kynurenic acid was similarly administered to normal rats (n=6), however, no changes were observed in MAP (Fig 3D⇑) or heart rate.
When proportional changes in blood pressure are compared between groups it is interesting to note that glycine administered into the RVLM caused a decrease in blood pressure with changes in MAP of 74.8±31.9 versus 46.4±12.5 mm Hg for 2K1C and control groups. After l-glutamate was administered the increase in blood pressure produced changes in MAP of 74.7±35.6 and 43.2±25.2 mm Hg, respectively.
The major new finding of this study was the demonstration that stimulation of the RVLM by l-glutamate in hypertensive rats caused a much higher increase in arterial pressure than observed in normotensive rats (228±34.9 versus 148.3±24.9 mm Hg), and that microinjection into the same area of the broad-spectrum antagonist kynurenic acid produced a long-lasting decrease in arterial pressure in rats with Goldblatt hypertension. When the same procedures were performed on normal rats, kynurenic acid microinjection into the RVLM did not change ABP, as previously reported.9 The mechanism involved in this response is not clear, but it is conceivable that in hypertensive rats there is a modification in the number and/or a change in the sensitivity of glutamatergic receptors in the RVLM following the development of hypertension, and these alterations may be involved in the generation and/or maintenance of high blood pressure in this model of hypertension.
The mechanisms by which the brain helps to propagate hypertension depend on the specific causes of the hypertensive disease. A substantial body of evidence suggests that elevation in sympathetic activity participates in the pathogenesis of hypertension. Sympathectomy and antihypertensive agents that work by blocking sympathetic transmission can interrupt the development and maintenance of experimental hypertension.1
Intrathecal administration of dl-2-amino-5-phosphonovaleric acid, an amino acid antagonist, reduced resting arterial pressure in spontaneously hypertensive rats and stroke-prone spontaneously hypertensive rats but not normotensive Wistar-Kyoto rats.10 Electrophysiological studies have confirmed increases in the firing rate of barosensitive neurons in the RVLM of spontaneously hypertensive rats.11 12 These results suggest an increase in the tonic activity of bulbospinal neurons containing excitatory amino acids, terminating in the intermediolateral cell column of the spinal cord in SHR rats. The increase in tonic glutamatergic activity in the intermediolateral cell column probably is caused by the RVLM.
In 2K1C rats we found that microinjection of kynurenic acid into the RVLM produced a decrease in ABP only in hypertensive rats and that microinjection of l-glutamate into the RVLM produced an impressive increase in ABP in hypertensive rats. In the latter case the glutamatergic activity of RVLM neurons is modified in 2K1C rats and may be involved in the increase of sympathetic activity observed in this model. There is a body of evidence that indicates that the sympathetic activity of intermediolateral column neurons is maintained by glutamatergic projections from the RVLM in both normotensive10 and spontaneously hypertensive rats.10 13 14 Our results suggest that there is a similar mechanism in 2K1C rats.
It is reasonable to suggest not only that the activity of RVLM neurons is important for the maintenance of high blood pressure in 2K1C rats but also that other areas of the nervous system may be involved in the generation and maintenance of the high sympathetic activity observed in this model. For example, since it has been shown that the anteroventral part of the third ventricle is a key area in the development of renovascular hypertension4 we may assume that projections from the anteroventral third ventricle to the RVLM may participate in the maintenance of elevated blood pressure in 2K1C animals; although the nature and characteristics of these projections remains to be determined, glutamate also may be involved.
The microinjection of glycine into the RVLM of hypertensive and normotensive rats produced a decrease in ABP. However, the ABP level observed after inhibition of RVLM in hypertensive rats was higher than that observed in normal rats, and a similar result was obtained with intravenous administration of hexamethonium. These results show not only that in the presence of hypertension the nervous system (ie, RVLM activity) is involved in the maintenance of arterial pressure but also that other mechanisms such as the renin-angiotensin system or vasopressin participate in this disturbance. The importance of the renin-angiotensin system in the initial development of renal hypertension is well established as also is the evidence for interactions between the renin-angiotensin system and sympathetic nervous system.1 4 Different results from our own were reached by Muratani et al,15 who observed a higher response in spontaneously hypertensive rats compared with control Wistar-Kyoto rats when these rats were treated with hexamethonium, emphasizing the difference between the model of Muratani et al and ours.
The heart rate alterations observed in our study were not significant for all groups, but use of anesthetized rats in the present set of experiments could have contributed to such alterations, as could the use of urethane, which alters the vagal tone. Anesthesia is a limiting factor in the baroreceptor modulation of arterial pressure alterations.
In summary, the present series of experiments shows that the activity of RVLM neurons is important for the maintenance of ABP in renovascular 2K1C hypertensive rats and that the glutamatergic activity of this area is involved in this model of hypertension.
Selected Abbreviations and Acronyms
|ABP||=||arterial blood pressure|
|MAP||=||mean arterial pressure|
|RVLM||=||rostral ventrolateral medulla|
|2K1C||=||two-kidney, one clip|
This work was supported by the Fundação de Amparo a Pesquisa do Estado de São Paulo/FAPESP (grant 91/0506/0).
- Received June 6, 1995.
- Revision received August 18, 1995.
- Accepted September 10, 1995.
Gordon F, Haywood R, Brody M, Johnson A. Effects of lesion of the AV3V on the development of hypertension in SHR. Hypertension. 1982;4:387-393.
Haywood R, Fink G, Buggy J, Boutelle S, Johnson A, Brody M. Prevention of 2K1C renal hypertension in rat by ablation of AV3V tissue. Am J Physiol. 1983;245:H683-H689.
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