Brain ‘Ouabain’ in the Median Preoptic Nucleus Mediates Sodium-Sensitive Hypertension in Spontaneously Hypertensive Rats
Pressor responses to an acute increase in cerebrospinal fluid sodium and exaggeration of the hypertension and sympathetic hyperreactivity in spontaneously hypertensive rats (SHR) by high sodium diet involve release of brain “ouabain” and subsequent activation of the brain renin-angiotensin system. In the present study, we determined whether release of “ouabain” in the median preoptic nucleus participates in these responses. In conscious Wistar rats, the pressor and heart rate responses to central hypertonic saline (0.3 mol/L NaCl, 3.8 μL/min over 10 minutes) and ouabain (0.6 μg) were compared after median preoptic nucleus injection of either γ-globulins or Fab fragments binding ouabain and brain “ouabain” with high affinity. Microinjection of Fab fragments into the median preoptic nucleus abolished the pressor and tachycardic responses to central hypertonic saline and significantly reduced the pressor response to central ouabain. In SHR on high sodium, microinjection of Fab fragments into the median preoptic nucleus significantly decreased baseline blood pressure to a level not different from that in SHR on regular sodium (149±7 versus 145±5 mm Hg), whereas the enhanced responses to air stress were not affected. Our results support the concept that blood pressure responses to central hypertonic saline and exaggeration of the hypertension in SHR by high sodium diet depend on release of brain “ouabain” in the median preoptic nucleus.
In SHR, high sodium intake exaggerates hypertension, accompanied by an increase in sympathetic activity and significant enhancement of sympathoexcitatory and pressor responses to stress.1 2 3 4 The mechanisms through which high sodium intake increases sympathetic activity are still not well established. We have postulated that consumption of a high sodium diet may result in intermittent increases in CSF sodium that lead to an increase in brain “ouabain” and activation of the brain renin-angiotensin system.1 5 6 Although discrepant results have been published regarding CSF sodium concentration in rats on high sodium, a significant increase in CSF sodium was observed when CSF samples represented light and night phases of the day.7 8 9 Moreover, intracerebroventricular infusion of hypertonic saline results in pressor responses that are mediated by release of brain “ouabain” and subsequent activation of the brain renin-angiotensin system.5 10 Studies in SHR on high sodium indicate that blockade of either brain “ouabain” or angiotensin II type 1 receptors prevents or reverses decreased sympathoinhibition and enhanced sympathoexcitation as well as the hypertension.6
The exact location of “ouabain” action in the CNS has not been established. Neuroanatomic studies showed “ouabain”-positive axons in various hypothalamic structures, including the lamina terminalis.11 12 This structure is considered to be a major center integrating cardiovascular and osmoregulatory responses.13 In particular, the MnPO is innervated by fibers coming from various brain structures, including A2 neurons in the nucleus of the solitary tract, the subfornical organ, and the organum vasculosum laminae terminalis.14 15 16 Besides a heavy innervation of the paraventricular nucleus, the MnPO also innervates various monoaminergic cell groups in the brain stem.15 17 Related to these connections arising from a variety of CNS structures, activity of afferents to this area may reflect the plasma concentration of fluid balance–associated hormones (eg, angiotensin II via connections from the subfornical organ) and activity of arterial, cardiopulmonary, and central osmoreceptors.13 14 15 On the other hand, efferent connections link this nucleus with cardiovascular regulatory and sympathoregulatory areas, providing a neuroanatomic basis for an integrative function of the MnPO. Moreover, lesions of the anteroventral third ventricle region (which includes the ventral part of the MnPO) prevent or attenuate several forms of experimental hypertension, including that in Dahl rats and deoxycorticosterone acetate–salt hypertensive rats.13 Lesion of the anteroventral third ventricle also impairs the natriuretic response to volume expansion, probably because of decreased release of Na+,K+-ATPase inhibitor.18
Thus, on the basis of anatomic and functional studies, we hypothesized that “ouabain” released in the MnPO mediates cardiovascular responses observed after intracerebroventricular administration of hypertonic saline and in sodium-dependent hypertension in SHR. Therefore, in the present experiments, we compared in conscious rats the pressor and HR responses to intracerebroventricular administration of hypertonic saline and exogenous ouabain before and after MnPO injection of Fab fragments, which bind ouabain and brain “ouabain” with high affinity.5 19 20 We also determined whether blockade of brain “ouabain” in the MnPO with these Fab fragments reverses the exaggeration of the hypertension and enhancement of cardiovascular responses to air stress in SHR on high sodium intake.
Experiments in series 1, 2, and 3 were performed in adult, male Wistar rats purchased from Charles River (Montreal, PQ, Canada). For experiments in series 4, young 3.5-week-old SHR (Taconic Farms, Germantown, NY) were used. The rats were housed under standard conditions (12-hour light cycle, ambient temperature of 23±2°C) and received standard laboratory chow or special diet and tap water ad libitum. After the rats arrived from the supplier, they were allowed up to 1 week to become accustomed to the new environment. All experimental procedures were approved and carried out in accordance with the guidelines of the University of Ottawa Animal Care Committee for the use and care of laboratory animals. Rat characteristics are presented in the Table⇓.
Implantation of Guide Cannulas
With rats under sodium pentobarbital (65 mg/kg IP) anesthesia, the dorsal surface of the skull was exposed, and the skull was leveled between bregma and lambda. Appropriate holes were drilled, and guide cannulas (0.7 mm in diameter) were implanted intracerebroventricularly and above the MnPO. The stereotaxic coordinates for the MnPO cannula were 0.4 mm caudal to the bregma, 1 mm lateral to midline, and 5.4 mm ventral from the surface of the skull at an angle of 10° in the coronal plane.21 The tip of the cannula remained 1 mm above the MnPO. The intracerebroventricular cannula was implanted 0.4 mm caudal to the bregma, 1.4 mm lateral to the midline, and 3 mm ventral from the surface of the skull. The cannulas were secured to the skull with two jeweler's screws and acrylic cement (HCG Hygenic Corp) and closed with stainless steel obturators, and rats were injected with 30 000 IU penicillin G IM (Longisil, Sanofi). Seven days were allowed for recovery.
Cannulation of Vessels
With rats under halothane anesthesia (2% halothane in 100% oxygen), Tygon catheters (Norton Performance Plastics Co) filled with heparin (1000 U/mL in 0.9% NaCl) were implanted in the abdominal aorta via a femoral artery and/or in the inferior vena cava via a femoral vein. The other end of the catheters was sealed, tunneled subcutaneously, exteriorized in the intrascapular region, and secured to the skin. Experiments started 24 hours after vessel cannulation. BP and HR were recorded with a Data Science International system. Briefly, the BP signal was transformed with a Statham transducer, amplified, and fed to the computer. Dataquest software allowed on-line analysis of the BP signal and data storage. Except for air stress data, where momentary changes in BP and HR were used, other individual measurements represent average values for BP and HR from 10-second periods.
Series 1 and 2: Blockade of Central Cardiovascular Action of Hypertonic Saline and Ouabain by Microinjection of Fab Fragments Into the MnPO
Series 1 and 2 experiments were performed in Wistar rats instrumented with MnPO and intracerebroventricular guide cannulas as well as arterial and venous catheters. On day 1, rats were connected to the BP-measuring unit, and MnPO and intracerebroventricular injection cannulas (0.25 mm in diameter) were inserted into the respective guide cannulas. Thirty minutes was allowed for stabilization of hemodynamic parameters. After baseline BP and HR had been recorded, the injection cannulas were advanced to their final positions so their lower tips extended 1 mm below the end of the guide cannulas. Then the MnPO was injected (intranuclear) with γ-globulins (Sigma Chemical Co; 17 μg in 250 nL aCSF) over 20 seconds, and 2 minutes later, the cannula was carefully removed. To ensure that the injection was successful, we monitored the flow of the injectate by the movement of a small air bubble that separated the injected solution from a vehicle in the catheter. Ten minutes later, the rats were injected intravenously with the vasopressin V1 antagonist d-(CH2)5-Tyr-(Me)AVP (30 μg/kg in 0.2 mL of 0.9% NaCl; Sigma). Five minutes after intravenous injection of the vasopressin antagonist, rats were infused intracerebroventricularly with hypertonic saline (0.3 mol/L NaCl, 3.8 μL/min) for 10 minutes. After the pressor response to intracerebroventricular hypertonic saline had subsided (approximately 20 minutes), the rats received intracerebroventricular injection of ouabain (Sigma; 0.6 μg in 5 μL aCSF). After the pressor response to intracerebroventricular ouabain had subsided, arterial and venous catheters were flushed with a solution of heparin in saline and sealed, and rats were returned to their home cage.
On day 2, the experimental procedure was repeated but instead of γ-globulins, Fab fragments (17 μg in 250 nL aCSF; Digibind, Glaxo-Wellcome) were injected into the MnPO. MnPO treatments in this protocol were not randomized because the time necessary for clearance of the Fab fragments from brain tissues is not well established and may be longer than 1 day. To exclude possible effects of “time” on the responses, in a separate experiment we established the reproducibility of the cardiovascular responses to hypertonic saline and ouabain. For this, we used the same protocol except for injections of γ-globulins on both day 1 and day 2.
To determine cardiovascular responses to volume injection into the cerebroventricular system, we performed a separate experiment (series 2) in Wistar rats implanted with intracerebroventricular guide cannulas and arterial catheters. Similar to series 1, they were infused with aCSF (3.8 μL/min ICV over 10 minutes) and 20 minutes later injected with 5 μL aCSF ICV while BP and HR were recorded.
Series 3: Cardiovascular Actions of Ouabain in the MnPO
As described in series 1, Wistar rats equipped with MnPO guide cannulas and arterial catheters were injected into the MnPO but this time with aCSF on day 1 and ouabain (100 ng in 250 nL aCSF) on day 2, and cardiovascular responses were recorded for 20 minutes. The treatments were not randomized to exclude the possibility that neurotoxic properties of ouabain22 would affect the responses to intranuclear injection of aCSF after previous injection of ouabain.
Series 4: Reversal of Sodium-Dependent Hypertension in SHR by MnPO Injection of Fab Fragments
Four days after SHR arrived from the supplier, they were randomly divided into two groups: a regular sodium intake group that received regular rat chow (101 μmol Na/g) and tap water and a high sodium intake group that received high sodium rat chow (1370 μmol Na/g; Harlan Sprague-Dawley, Inc) and tap water.
The rats were weighed weekly. After 4 weeks of diet, an MnPO guide cannula was implanted as described above. After 1 week of recovery, rats were implanted with arterial catheters. On the next day at the beginning of the experiment, the rats were placed in small cages that allowed for back-and-forth movement. After 30 to 60 minutes of stabilization, baseline BP and HR were recorded. Then a standardized air stress (jet of air at 1.5 PSI) lasting 30 seconds was provided twice, with a 10-minute interval between the two stresses. Next, similar to series 1, rats on both diets received random injections into the MnPO of either γ-globulins (34 μg/250 nL aCSF) or Fab fragments (34 μg/250 nL aCSF). Two hours later, measurements of baseline BP and HR as well as air stress were repeated. Subsequently, the arterial catheter was flushed with heparinized saline, disconnected, and sealed, and rats were allowed to rest. On the next day, 18 hours after intranuclear injection, the arterial catheter was connected to a pressure transducer, and after a 30- to 60-minute stabilization period, BP and HR were recorded, and air stress was provided.
Verification of Cannula Position
At the end of the experiment, rats were deeply anesthetized with sodium pentobarbital (100 mg/kg IP) and injected with 250 nL of 25% India ink solution into the MnPO and with 5 μL intracerebroventricularly where applicable. Subsequently, rats were perfused transcardially with 150 mL of 10 mmol/L phosphate-buffered saline followed by 200 mL of 4% paraformaldehyde. The brains were removed and postfixed in the same fixative overnight at 4°C. On the next day, the brains were cut on blocks and inspected for staining of the cerebroventricular system in series in which an intracerebroventricular cannula was inserted. In the experiments in which an MnPO cannula was implanted, 50-μm-thick sections were cut through the forebrain on a vibratome and mounted on gelatinized slides. Then the sections were stained with 2% cresyl violet for determination of the injection site. Only rats properly injected in the MnPO and with an intracerebroventricular cannula in the lateral ventricle were used for statistical analysis.
All data are presented as mean±SE. One-way or factorial ANOVA was applied where appropriate.23 Individual differences were isolated with Newman-Keuls test. When air stress was applied, an average of two BP and HR responses was used for analysis. Differences were considered significant at a value of P<.05.
Blockade of Cardiovascular Effects of Central Hypertonic Saline and Ouabain
In normotensive Wistar rats, intranuclear injection into the MnPO of either γ-globulins or Fab fragments did not change resting MAP (−1±2 versus −2±1 mm Hg, P>.05) and HR. Intracerebroventricular infusion of aCSF resulted in minor nonsignificant changes in MAP and HR (Fig 1⇓). In control conditions (intranuclear γ-globulins), intracerebroventricular infusion of hypertonic saline significantly increased MAP and HR. Injection of Fab fragments into the MnPO reduced these pressor and HR responses to a level not different from levels in volume control experiments (Fig 1⇓).
In control conditions (intranuclear γ-globulins), intracerebroventricular injection of 0.6 μg ouabain caused significant increases in MAP and HR. When Fab fragments were injected into the MnPO, the increase in MAP was significantly reduced by 60% (Fig 2⇓).
When rats (n=7) were injected twice with γ-globulins, administration of 0.3 mol/L NaCl and ouabain caused comparable MAP and HR responses on both days (for NaCl infusion, 21±2 versus 18±3 mm Hg and 41±10 versus 38±12 beats per minute; for ouabain, 17±3 versus 17±6 mm Hg and 33±14 versus 40±8 beats per minute on day 1 versus day 2).
Cardiovascular Actions of Ouabain in the MnPO
Injection of aCSF into the MnPO did not change MAP or HR. In contrast, injection of 100 ng ouabain resulted in a significant increase in MAP, whereas HR did not increase significantly (Fig 3⇓).
Reversal of Sodium-Dependent Hypertension in SHR
Baseline MAP and HR
Five weeks of high sodium diet significantly exacerbated hypertension in SHR (Table⇑). In SHR on regular sodium, injection of γ-globulins and Fab fragments did not change MAP and HR. In contrast, in SHR on high sodium, intranuclear injection of Fab fragments resulted in a gradual decrease in MAP. The maximal decrease in MAP was observed 90 minutes to 2 hours after intranuclear injection (data not shown). After 2 hours, this decrease was significantly different from changes observed in SHR on high sodium after γ-globulins and in SHR on regular sodium after Fab fragment injection (Fig 4⇓). After intranuclear injection of Fab fragments, MAP in SHR on high sodium was no longer different from baseline MAP in SHR on regular sodium (149±7 versus 145±5 mm Hg). The decrease in MAP after MnPO Fab fragments in SHR on high sodium gradually subsided and at 18 hours after the injection was no longer statistically different from results with other treatments (Fig 4⇓). In contrast to MAP, HR was not influenced by MnPO injection of either γ-globulins or Fab fragments in any of the groups.
Air Stress Responses
SHR on high sodium responded to air stress with significantly higher increases in MAP and HR than SHR on regular sodium (Fig 5⇓). Intranuclear injection of γ-globulins or Fab fragments did not change responses to air stress in any of the groups.
The present study indicates that in conscious, normotensive rats, release of brain “ouabain” in the MnPO mediates the pressor responses to an increase in CSF sodium and participates in the exaggeration of hypertension in SHR by high sodium intake.
Central Cardiovascular Actions of Hypertonic Saline and Ouabain
Injection of Fab fragments into the MnPO did not exert any appreciable changes in MAP and HR, indicating that under resting conditions, “ouabain” is probably not released in amounts sufficient to exert significant effects. On the other hand, the MnPO appears to mediate responses to exogenous ouabain applied intracerebroventricularly. Microinjection of Fab fragments into the MnPO significantly reduced pressor but not HR responses to intracerebroventricular ouabain. Intracerebroventricular administration of Fab fragments abolished increases in BP, HR, and renal sympathetic activity in response to intracerebroventricular ouabain.5 Although the amount of Fab fragments applied in the previous study5 was four times higher than in the present study, it is rather unlikely that lack of total abolishment of the responses results from an insufficient amount of antagonist. With the different volume of distribution taken into consideration, the tissue concentration of Fab fragments in the present study was more likely to be higher, providing high binding capacity for ouabain. Since ouabain may bind to virtually every cell in the CNS, it is likely that the pressor and HR effects of intracerebroventricular ouabain may result from activation of various CNS nuclei. Therefore, intracerebroventricular administration of Fab fragments, which allows for penetration to a variety of CNS structures, can totally block the responses. Microinjection of ouabain into the MnPO caused a pressor response that was accompanied by an insignificant increase of HR. This finding is in agreement with the study by Jones and Lo24 in which ouabain injected into the MnPO of anesthetized normotensive Wistar rats also exerted pressor but not significant tachycardic responses.
The hypertonic saline experiment suggests that in the MnPO, “ouabain” may play an important role in BP regulation. Results from this experiment show that the MnPO is a structure in which ouabain exerts its action under physiological/pathophysiological conditions, since pressor and HR responses to intracerebroventricular hypertonic saline were almost abolished by Fab fragment injection into the MnPO. Intracerebroventricular infusion of hypertonic saline is known to increase BP, HR, and renal sodium excretion.5 25 26 27 These responses are accompanied by a variety of neurohumoral changes, including activation of the sympathetic nervous system, release of vasopressin and corticotropin, and suppression of the peripheral renin-angiotensin system.5 28 29 In previous experiments, we provided evidence that increases in MAP, HR, and renal sympathetic activity in response to intracerebroventricular hypertonic saline result first from central release of “ouabain” and then activation of the brain renin-angiotensin system.5 10 Intracerebroventricular infusion of hypertonic saline after isolation of the anterior portion of the third ventricle from the cerebroventricular system demonstrated that structures adjacent to this area are crucial for the occurrence of the pressor response.30 The present study provides new evidence that virtually the entire pressor response to hypertonic saline depends on activation of the MnPO. Since the tachycardia in Fab experiments was not different from that in volume control experiments, it also indicates that not only the pressor but also the tachycardic response to intracerebroventricular hypertonic saline depend on the release of “ouabain” in the MnPO. Interestingly, in contrast to administration of exogenous ouabain, release of “ouabain” in the MnPO seems to also affect HR-regulating mechanisms. It is possible that an increase in CSF Na+ results in release of other neurotransmitters in the MnPO that modify the action of “ouabain.” Such interaction between catecholamines and angiotensin II with regard to its pressor and dipsogenic effects has already been reported.14 31 Our data further confirm that the MnPO may be a key structure linking changes in extracellular osmolality or sodium concentration and regulation of the cardiovascular system. Furthermore, they point to “ouabain” as the mediator in this process.
Reversal of Sodium-Dependent Hypertension in SHR
The present study demonstrates that release of “ouabain” in the MnPO plays a key role in the exaggeration of the hypertension in SHR by high sodium. Blockade of brain “ouabain” in the MnPO by localized injection of Fab fragments reversed the hypertension caused by high sodium in SHR. Interestingly, it did not normalize MAP and HR responses to air stress.
In previous studies from this laboratory, intracerebroventricular injection of Fab fragments abolished exaggeration of the hypertension in SHR by high sodium. Moreover, Fab fragments also normalized MAP responses to intracerebroventricular guanabenz and air stress.6 Similarly, in Dahl salt-sensitive rats on high sodium, blockade of brain “ouabain” reversed hypertension and normalized responses to guanabenz and air stress.32 The present study indicates that the MnPO mediates the hypertensive action of increased dietary sodium, consistent with studies showing that the MnPO and other structures located in the anteroventral border of the third ventricle participate in osmoregulation and control of sympathetic activity (for review, see Reference 13). Furthermore, these results are consonant with results from the hypertonic saline experiment, which indicates that the MnPO mediates the pressor response to an acute increase in CSF sodium. Despite the important role of this area in the regulation of sympathetic outflow, it appears that the enhancement of cardiovascular responses to air stress by high sodium does not depend on the integrity of this structure. Hatton et al33 reported that destruction of the anteroventral third ventricle, which also contains the MnPO, in borderline hypertensive rats on 1% NaCl diet did not change BP and HR responses to air stress. The present study indicates that enhanced pressor responses to air stress in SHR on high sodium do not appear to be critically dependent on the release of “ouabain” in the MnPO. As mentioned above, increased pressor, HR, and renal sympathetic nerve activity responses to air stress in SHR on high sodium and Dahl salt-sensitive rats could be reversed by intracerebroventricular administration of Fab fragments.6 32 In the present study, lack of normalization of MAP and HR responses to air stress despite a decrease in baseline BP indicated that enhanced responsiveness in SHR on high sodium did not result from the higher BP but represented a separate mechanism. Moreover, it appears that other populations of “ouabainergic” neurons distinct from the MnPO neurons mediate the enhancement of cardiovascular responses to air stress. Various brain nuclei and transmitters have been postulated to mediate cardiovascular responses to environmental stress in hypertensive and normotensive rats.34 35 36 So far, no report has demonstrated involvement of any brain nuclei in the potentiation of sympathoexcitatory responses to air stress by high sodium. It can only be speculated that this structure could be part of the limbic-hypothalamic circuitry.37
In conclusion, our results support the concept that increases in BP observed in normotensive rats after acute increases in CSF sodium and the exaggeration of the hypertension in SHR by high sodium diet depend on release of brain “ouabain” in the MnPO.
Selected Abbreviations and Acronyms
|aCSF||=||artificial cerebrospinal fluid|
|CNS||=||central nervous system|
|MAP||=||mean arterial pressure|
|MnPO||=||median preoptic nucleus|
|SHR||=||spontaneously hypertensive rat(s)|
This work was supported by an operating grant from the Medical Research Council of Canada. A.S.B. is a postdoctoral fellow from the Department of Clinical and Applied Physiology, Warsaw School of Medicine, supported by a Research Fellowship from the Heart and Stroke Foundation of Ontario. F.H.H.L. is a Career Investigator of the Heart and Stroke Foundation of Ontario. We thank Glaxo Wellcome, Toronto, Canada, for generously providing Digibind.
Reprint requests to Frans H.H. Leenen, MD, PhD, FRCP(C), Hypertension Unit, University of Ottawa Heart Institute, 40 Ruskin St, Ottawa, Ontario, Canada K1Y 4E9. E-mail email@example.com
- Received June 3, 1996.
- Revision received July 19, 1996.
- Revision received September 18, 1996.
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