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Am J Physiol Heart Circ Physiol 274: H1269-H1276, 1998;
0363-6135/98 $5.00
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Vol. 274, Issue 4, H1269-H1276, April 1998

Brain "ouabain," ANG II, and sympathoexcitation by chronic central sodium loading in rats

Bing S. Huang, Shereeni J. Veerasingham, and Frans H. H. Leenen

University of Ottawa Heart Institute, Ottawa, Ontario K1Y 4E9, Canada

    ABSTRACT
Top
Abstract
Introduction
Methods
Results
Discussion
References

Both brain ouabain-like activity ("ouabain") and brain angiotensin II (ANG II) contribute to the sympathoexcitatory and pressor responses to high sodium intake in spontaneously hypertensive (SHR) and Dahl salt-sensitive (Dahl S) rats. To assess whether increases in cerebrospinal fluid (CSF) sodium can mimic this pattern of changes, Wistar rats were chronically infused with artificial CSF (aCSF) or sodium-rich aCSF (0.8 or 1.2 M sodium) intracerebroventricularly through osmotic minipumps for 14 days. Sodium-rich aCSF (0.8 M) was also infused intracerebroventricularly for 2 wk concomitantly with either antibody Fab fragments that bind ouabain and related steroids with high affinity, gamma -globulins as control (200 µg/day for both), or the AT1 blocker losartan (1 mg · kg-1 · day-1). Sodium-rich aCSF increased CSF sodium from 146 ± 2 to 152 ± 2 (0.8 M) and 160 ± 3 (1.2 M) mmol/l, and increased brain "ouabain" in the hypothalamus, pituitary, and pons. In conscious rats, sodium-rich aCSF increased baseline mean arterial pressure (MAP), enhanced MAP, heart rate (HR), and renal sympathetic nerve activity (RSNA) responses to intracerebroventricular alpha 2-adrenoceptor agonist guanabenz and air stress, and desensitized arterial and cardiopulmonary baroreflex control of HR and RSNA. These effects were largely prevented by intracerebroventricular Fab fragments or losartan. Thus, in Wistar rats, both brain "ouabain" and the brain renin-angiotensin system contribute to sympathoexcitation, impairment of baroreflexes, and hypertension caused by chronically increased CSF sodium. The similar patterns of changes caused by CSF sodium in Wistar rats and by high sodium intake in SHR and Dahl S rats indicate that if high sodium intake increases central sodium, such changes may contribute to sympathoexcitation and hypertension.

cerebrospinal fluid sodium; baroreflex; sympathetic nerve activity; guanabenz

    INTRODUCTION
Top
Abstract
Introduction
Methods
Results
Discussion
References

IN RATS GENETICALLY predisposed to hypertension, i.e., spontaneously hypertensive (SHR) and Dahl salt-sensitive (Dahl S) rats, high sodium intake exacerbates the development of hypertension, which to a large extent appears to result from increased sympathetic outflow (30, 33). In both SHR and Dahl S rats, high dietary sodium causes decreased sympathoinhibition, enhanced sympathoexcitation, and impairment of arterial as well as cardiopulmonary baroreflex control of sympathetic nerve activity (13-18). Brain ouabain-like activity ("ouabain") appears to play a major role in these central effects of high dietary sodium (13-16, 18) and to cause sympathoexcitation and hypertension by activating the brain renin-angiotensin system (19). It has not yet been elucidated how increased dietary sodium increases brain "ouabain." We proposed (19) that in sodium-sensitive rats increased dietary sodium elevates cerebrospinal fluid (CSF) sodium transiently or intermittently, and increased brain sodium increases synthesis and release of brain "ouabain," leading to sympathoexcitation, impairment of baroreflexes, and hypertension.

Acute (2, 17) and chronic (3) intracerebroventricular infusions of NaCl cause sympathetic hyperactivity and hypertension in normotensive rats. Chronic intracerebroventricular sodium loading also desensitized the arterial baroreflex control of heart rate (HR) but not renal sympathetic nerve activity (RSNA) in normotensive rats under general anesthesia (3). The effects of acute intracerebroventricular sodium are mediated via brain "ouabain" (17), and the latter induces sympathoexcitation likely via brain angiotensin II (ANG II) (17). At present, it is unknown whether in normotensive rats a chronic increase in CSF sodium can mimic the pattern of central changes caused by high sodium intake in Dahl S rats and SHR (13, 19), i.e., whether increased CSF sodium increases brain "ouabain," and if so, whether increased brain "ouabain" and increased brain ANG II receptor stimulation are responsible for the sympathetic hyperactivity, desensitization of baroreflexes, and the hypertension.

Hence, in the present study, we examined in Wistar rats 1) the effects of intracerebroventricular sodium-rich (0.8-1.2 M) artificial CSF (aCSF) for 2 wk on CSF sodium concentration, brain "ouabain," and resting blood pressure (BP), as well as sympathoexcitatory and pressor responses and arterial and cardiopulmonary baroreflex control of RSNA and HR and 2) whether concomitant chronic blockade of brain "ouabain" or ANG II can prevent the sympathoexcitation, impairment of baroreflex function, and hypertension caused by the central sodium loading. All functional studies were performed in conscious state to exclude confounding effects of general anesthesia (7). The results indicate that Wistar rats with a chronic increase in CSF sodium do mimic the pattern of central changes caused by high dietary sodium intake in SHR and Dahl S rats.

    METHODS
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Abstract
Introduction
Methods
Results
Discussion
References

Male Wistar rats (Charles River, Montreal, Canada) weighing 150-200 g were used. They were housed individually at constant room temperature and illuminated from 6 AM to 6 PM. Regular laboratory rat chow and tap water were provided ad libitum. All experimental procedures were carried out according to the guidelines of the University of Ottawa Animal Care Committee.

Protocol 1

After 3 days of acclimatization, under pentobarbital sodium (65 mg/kg ip) anesthesia, a stainless steel right-angled cannula was implanted into the left lateral ventricle of the brain as described previously (13). Coordinates were 0.5 mm posterior and 1.4 mm lateral to bregma. The lower end of the cannula was at a depth of 3.5 mm from dura, and the upper end was connected to an osmotic minipump (model 2ML2, Alza, Palo Alto, CA) for chronic intracerebroventricular infusion at 5 µl/h for 14 days. Considering the secretion rate of CSF in rats (120-320 µl/h) (6), this low rate of intracerebroventricular infusion unlikely affects CSF pressure. The pumps were filled with aCSF (n = 8), aCSF containing 0.8 M NaCl (n = 9), or aCSF containing 1.2 M NaCl (n = 9), and implanted subcutaneously on the back of the rats. The animals were then returned to their original cage with regular food and water. They were trained to stay quietly in a small experimental cage (24 × 15 × 8 cm) in which the rat can move back and forth on three to four different occasions, each lasting 1-2 h.

On day 14, under halothane anesthesia, a PE-50 polyethylene catheter was positioned in the right femoral artery. The catheter was filled with heparin, sealed with a stylet, tunneled subcutaneously, and exteriorized on the back of the neck. On day 15, the conscious rat was placed in the experimental cage. The catheter was connected to a Grass 7P511 amplifier, a Grass 7P44 tachograph, as well as a Grass polygraph (model 7E) through Statham P23 1D transducer, for BP and HR recording. After a 30-min rest, baseline mean arterial pressure (MAP) and HR were recorded for 3 min. A standardized air stress was then provided twice at a 10-min interval by blowing the face of the rat with a jet of air (1-1.5 psi) for 30 s, from a tube located approx 3 cm in front of the rat. The average of peak changes in MAP and HR in response to two application of stress was used for comparisons. The animals were killed with an overdose of pentobarbital, and the whole brain and pituitary were removed. The tissue samples were stored at -70°C. At the time of assay the hypothalamus and pons were dissected at 4°C according to Glowinski and Iversen (8). The preparation of tissue extracts and the quantitation of "ouabain" by estimating its inhibitory effect on Na+-K+-adenosinetriphosphatase (ATPase) activity were described previously (22).

Measurement of CSF sodium. In a different group of 15 rats with chronic intracerebroventricular cannulation, aCSF or aCSF containing 0.8 or 1.2 M NaCl (n = 5 for each) was randomly infused intracerebroventricularly for 14 days, as previously described. On day 15, BP and HR were recorded, and 1 ml of blood was withdrawn from the arterial line in conscious rats. Physiological saline (1 ml) was injected intravenously for replacing the blood sample. Subsequently, under pentobarbital sodium anesthesia, each rat was placed in a stereotaxic frame, and 100-200 µl clear CSF sample was collected at <5 µl/s from a cannula that was inserted into the cisterna magna, as described previously (3). Sodium and potassium concentrations were determined in all plasma and CSF samples by using an ion-selective electrode (Hitachi electrode, model 917) in the Department of Laboratory Medicine, University of Ottawa Civic Hospital.

Protocol 2

The same brain surgery as in protocol 1 was performed. The rats were randomly divided into four groups: 1) aCSF containing gamma -globulins (Sigma Chemical, St. Louis, MO; n = 7); 2) aCSF containing 0.8 M NaCl plus gamma -globulins (n = 7); 3) aCSF containing 0.8 M NaCl plus antibody Fab fragments (Digibind, Glaxo Wellcome, Toronto, Canada; n = 7); and 4) aCSF containing 0.8 M NaCl plus AT1 receptor blocker losartan (DuPont Pharmaceuticals, Wilmington, DE; n = 8). The infusion rate of the pump was 5 µl/h for all. The Fab fragments or gamma -globulins and losartan were dissolved to obtain rates of infusion at 200 µg/day and 1 mg · kg body wt-1 · day-1, respectively. These rates prevent the sympathoexcitatory and pressor effects of high sodium intake in SHR and Dahl S rats (13, 18, 19), whereas intravenous administrations of Fab fragments as well as losartan at the same rates is ineffective (19). Intracerebroventricular losartan at higher doses (10 mg · kg-1 · day-1) also causes peripheral AT1 receptor blockade (21). With use of the same coordinates, a 23-gauge straight stainless steel cannula was subsequently implanted and fixed to the skull, serving as a guide cannula for acute intracerebroventricular injection, with its lower end about 0.5 mm above right lateral ventricle. Penicillin G (30,000 IU, Derapan, Ayerst Lab, Montreal, Canada) was given intramuscularly after the surgery. As in protocol 1, the rats were trained to stay quietly in the experimental cage.

At the end of the 2-wk infusion period, under halothane inhalation, catheters were placed in the right femoral artery and vein. A third catheter was inserted into the right jugular vein with its tip advanced to the level of right atrium, for the measurement of central venous pressure (CVP). With supplemental methohexital sodium (30 mg/kg iv, Brevital, Eli Lilly Canada, Toronto, Canada), a pair of silver electrodes (A-M System, Everett, WA) was placed around the left renal nerve through a flank incision and secured with silicone rubber (Wacker, Munich, Germany) (13). The electrodes and catheters were exteriorized at the back of the neck.

At least 4 h after the surgery the rat was placed in the experimental cage. The catheters and electrodes were connected to the Grass polygraph and a Grass P511 band-pass amplifier, for BP, CVP, HR, and RSNA recording. RSNA (spikes/s) was counted through a nerve traffic analyzer (model 706C, University of Iowa Bioengineering, Iowa City, IA). The actual nerve activity was determined by subtracting noise from the total activity. The background noise was measured by recording the activity 20 min after the rats had been killed by an overdose of pentobarbital at the end of each experiment (13).

After a 30-min rest, baseline MAP, HR, CVP, and RSNA were recorded for 3 min. Air stress was provided twice at a 10-min interval as described in protocol 1.

After a 15-min rest phenylephrine (5-50 µg/min) dissolved in normal saline was infused intravenously to achieve a ramp increase in MAP with a maximum of 50 mmHg over 2-3 min. After the responses had subsided and with an additional 15-min stabilization period, nitroprusside (5-100 µg/min) was infused intravenously, inducing a ramp decrease in MAP with a maximum of -50 mmHg. Infusion rates were less than 0.08 ml/min. MAP, HR, and RSNA returned to baseline levels within 5 min after the termination of infusions.

Subsequently, after a 15-min rest, acute volume expansion was performed through intravenous 5% dextrose at two rates (3.3 and 10.0 ml · kg body wt-1 · 30 s-1) at a 10-min interval. Intravenous infusions were accomplished with a Sage 355 infusion pump.

The animal was then allowed to rest for 30 min. With the use of a Hamilton microsyringe (10-µl volume), guanabenz (25 and 75 µg · 1-3 µl aCSF-1 · 10 s-1) was administered intracerebroventricularly at a 15-min interval through a 26-gauge stainless steel needle, which was inserted into the guide cannula so that its tip protruded into the lateral ventricle.

Data analysis. Responses of RSNA were expressed as percent changes from the baseline levels. To assess arterial baroreflex function, the percent changes of RSNA (Delta RSNA, %baseline) or changes of HR (Delta HR, beats/min) in response to MAP were analyzed as a logistic model (11) using the logistic equation: Delta RSNA = P1 + P2 /[1 + eP3(MAP - P4)], where P1 is lower Delta RSNA plateau, which represents the maximum decrease in RSNA, P2 is Delta RSNA range, P3 is a curvature coefficient, and P4 is MAP50, i.e., the MAP at half the Delta RSNA range. The upper plateau equals P1 + P2, which is the maximum increase in RSNA. The curve of best fit was obtained via a computer program provided by the Baker Medical Research Institute (Victoria, Australia). When calculated maximum decreases in RSNA were less than -100%, -100% was used as the maximum decrease for statistical analysis. The cardiopulmonary baroreflex function was evaluated by means of the gain of the reflex, which is the slope of the linear relations assessed by plotting Delta RSNA or Delta HR against corresponding CVP and analyzing it with linear regression. Because gains for the two volume expansion rates were similar, data from the two were pooled together for analysis. Two-way analysis of variance was performed for the comparisons. When F ratios were significant, a Duncan multirange test was followed. Statistical significance was defined as P < 0.05.

    RESULTS
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Abstract
Introduction
Methods
Results
Discussion
References

Protocol 1

Compared with rats with aCSF, in rats with 0.8 and 1.2 M NaCl, the content of brain "ouabain" was significantly higher in all three brain regions assessed (Table 1). The increases were more pronounced in rats treated with 1.2 vs. 0.8 M NaCl (Table 1). Compared with rats treated with intracerebroventricular aCSF, in rats treated with 0.8 and 1.2 M NaCl the baseline MAP was significantly elevated, and HR tended to be increased (Table 1). There were no significant differences of body weight gain among the three groups of rats (not shown).

                              
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  		Table 1.   Brain "ouabain" content and MAP and HR at rest and in response to air stress in rats treated with aCSF or aCSF containing 0.8 or 1.2 M NaCl for 14 days

Air stress increased MAP and HR, and the peak increases in both MAP and HR in rats treated with 0.8 or 1.2 M NaCl were significantly larger vs. those in rats with aCSF (Table 1).

In rats infused with 0.8 M NaCl, CSF sodium was significantly higher vs. that in rats treated with aCSF alone; 1.2 M NaCl intracerebroventricularly further increased CSF sodium, compared with 0.8 M NaCl intracerebroventricularly (Table 2). CSF potassium as well as plasma sodium and potassium concentrations were not significantly different among the three groups of rats (Table 2).

                              
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  		Table 2.   Plasma and CSF sodium and potassium concentrations and resting MAP and HR in rats infused intracerebroventricularly with aCSF or aCSF containing 0.8 or 1.2 M NaCl for 14 days

Protocol 2

Baseline data. At the end of the 2 wk of intracerebroventricular infusion in rats treated with 0.8 M NaCl and gamma -globulins baseline MAP and HR were significantly higher vs. those in rats treated with aCSF plus gamma -globulins (Table 3). The increases in BP and HR by 0.8 M NaCl were prevented by concomitant intracerebroventricular Fab fragments or losartan. There were no significant differences in baseline CVP and the gain of body weight.

                              
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  		Table 3.   Resting MAP, HR, CVP, and gain of body wt in rats after treatment with intracerebroventricular aCSF plus gamma -globulins or 0.8 M NaCl plus gamma -globulins, Fab fragments, or losartan for 14 days

Responses to air stress. Air stress increased BP, HR, and RSNA (Fig. 1). Peak increases in MAP, HR, and RSNA were significantly larger in rats treated with 0.8 M NaCl plus gamma -globulins vs. aCSF plus gamma -globulins. These increases in peak responses were not present when Fab fragments or losartan was infused intracerebroventricularly concomitantly with 0.8 M NaCl.


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  		Fig. 1.   Peak increases in mean arterial pressure (MAP), renal sympathetic nerve activity (RSNA), and heart rate (HR) in response to air stress. Values are means ± SE (for n see Table 2). * P < 0.05 vs. other groups. aCSF, artificial cerebrospinal fluid. H-Na, 0.8 M NaCl in aCSF; Fab, antibody Fab fragments; los, losartan.

Responses to guanabenz. Intracerebroventricular injection of guanabenz decreased MAP, HR, and RSNA in a dose-related manner (Fig. 2). Peak decreases were significantly larger in rats treated with 0.8 M NaCl plus gamma -globulins vs. aCSF plus gamma -globulins. These enhanced decreases in MAP, HR, and RSNA by 0.8 M NaCl were prevented when Fab fragments or losartan was intracerebroventricularly infused together with 0.8 M NaCl.


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  		Fig. 2.   Peak decreases in MAP, RSNA, and HR in response to intracerebroventricular injection of 25 (open bars) and 75 (hatch bars) µg of guanabenz. Values are means ± SE (for n see Table 2). * P < 0.05 vs. other groups. H-Na, 0.8 M NaCl in aCSF; Fab, antibody Fab fragments; los, losartan.

Arterial baroreflex. Ramp increases or decreases in BP elicited gradual decreases or increases in RSNA and HR (Figs. 3 and 4). Compared with rats treated with aCSF plus gamma -globulins, in rats infused with 0.8 M NaCl plus gamma -globulins the maximal increase in RSNA, as reflected by the first plateau of the Delta RSNA-MAP reflex curve, was significantly attenuated (Table 4). Moreover, the maximum slope as well as the slope between 25 and 75% of RSNA response range of the curve, were significantly less in rats with 0.8 M NaCl plus gamma -globulins vs. rats with aCSF plus gamma -globulins (Table 4), indicating an impaired arterial baroreflex control of RSNA in rats treated with 0.8 M NaCl. In rats treated with 0.8 M NaCl plus Fab fragments or losartan, the extent of maximum increase in RSNA and the slopes of reflex control were significantly increased vs. those in rats with 0.8 M NaCl plus gamma -globulins, and similar to those in rats treated with aCSF plus gamma -globulins (Fig. 3 and Table 4).


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  		Fig. 3.   Arterial baroreflex control of RSNA analyzed as logistic model. Each point represents mean ± SE of changes in RSNA (%baseline) relative to MAP changed by intravenous infusion of nitroprusside or phenylephrine (for n see Table 2). aCSF, artificial cerebrospinal fluid plus gamma -globulins; HNa, 0.8 NaCl in aCSF plus gamma -globulins; HNa + Fab, 0.8 M NaCl in aCSF plus Fab fragments; HNa + los, 0.8 M NaCl in aCSF plus losartan.


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  		Fig. 4.   Arterial baroreflex control of HR analyzed as logistic model. Each point represents mean ± SE of changes in HR (beats/min) relative to MAP changed by intravenous infusion of nitroprusside or phenylephrine (for n see Table 2). aCSF, artificial cerebrospinal fluid plus gamma -globulins; HNa, 0.8 NaCl in aCSF plus gamma -globulins; HNa + Fab, 0.8 M NaCl in aCSF plus Fab fragments; HNa + los, 0.8 M NaCl in aCSF plus losartan.

                              
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  		Table 4.   Estimated parameters of arterial baroreflex in rats after treatment with intracerebroventricular aCSF plus gamma -globulins, or 0.8 M NaCl plus gamma -globulins, Fab fragments, or losartan for 14 days

Compared with rats infused with aCSF, in rats treated with 0.8 M NaCl plus gamma -globulins the extent of maximum increase of HR (Fig. 1), maximum slope, and the slope between the 25-75% HR response (Table 4) were all significantly decreased. These effects of 0.8 M NaCl on the maximum increase in HR and the slopes did not develop when Fab fragments or losartan was concomitantly administered intracerebroventricularly (Fig. 4 and Table 4).

Cardiopulmonary baroreflex. Volume expansion at both rates significantly increased CVP and decreased RSNA and HR (Figs. 5 and 6). Volume expansion at the end of the higher rate caused a minor increase (<3 mmHg, not significant) in MAP. For the same rates of volume expansion, peak increases in CVP were similar for the four groups. However, the extent of maximum decrease in RSNA or HR in rats with 0.8 M NaCl plus gamma -globulins was significantly less than in other groups (Figs. 5 and 6). Moreover, in rats treated with 0.8 M NaCl plus gamma -globulins vs. rats with aCSF, the gain of Delta RSNA was significantly decreased (-8.8 ± 0.6 vs. -10.6 ± 0.7% baseline/mmHg, P < 0.05). In rats with 0.8 M NaCl plus Fab fragments or losartan this blunting was absent (-11.6 ± 1.0 or -11.2 ± 0.9 vs. -10.6 ± 0.7% baseline/mmHg, not significant for both). The differences for the gain of Delta HR vs. CVP followed a similar pattern as Delta RSNA (Fig. 6) but did not reach statistical significance (-6.1 ± 0.8 vs. -7.2 ± 0.6, -7.8 ± 0.8, or -7.7 ± 0.6 beats · min-1 · mmHg-1, P = 0.07-0.09).


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  		Fig. 5.   Changes in RSNA relative to central venous pressure (CVP) increased by volume expansion in rats with aCSF plus gamma -globulins (aCSF), aCSF containing 0.8 M NaCl plus gamma -globulins (HNa), aCSF containing 0.8 M NaCl plus Fab fragments (HNa + Fab), and aCSF containing 0.8 M NaCl plus losartan (HNa + los). Each point is mean ± SE of RSNA (%baseline) response at corresponding CVP (mmHg; for n see Table 2).


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  		Fig. 6.   Changes in HR relative to CVP elevated by volume expansion in rats with aCSF plus gamma -globulins (aCSF), aCSF containing 0.8 M NaCl plus gamma -globulins (HNa), aCSF containing 0.8 M NaCl plus Fab fragments (HNa + Fab), and aCSF containing 0.8 M NaCl plus losartan (HNa + los). Each point is mean ± SE of HR (beats/min) response at corresponding CVP (mmHg; for n see Table 2).

    DISCUSSION
Top
Abstract
Introduction
Methods
Results
Discussion
References

The present study provides as major new findings that 1) in conscious normotensive rats intracerebroventricular sodium for 14 days results in sodium "dose"-related increases in CSF sodium and brain "ouabain" in several brain areas, causes sympathetic hyperreactivity and impairment of arterial and cardiopulmonary baroreflex function, and increases resting BP; and 2) blockade of brain "ouabain" or brain ANG II prevents the sympathoexcitatory and hypertensive effects as well as impairment of baroreflexes caused by a chronic increase in CSF sodium.

Sympathoexcitation and Hypertension

In the present study infusion of 0.8 or 1.2 M NaCl at 5 µl/h into the left lateral ventricle for 14 days significantly increased CSF sodium at the cisterna magna by 6-14 mmol/l and baseline MAP by 19-25 mmHg. These results are consistent with studies by other groups (3, 20) in which 0.8 M NaCl for 11 days or 1.5 M NaCl for 7 days (both at 5.5 µl/h icv) increased CSF sodium by 8 or 5 mmol/l and MAP by 12 or 15 mmHg, respectively. After the development of hypertension by central sodium loading, ganglionic blockade (20) but not intravenous vasopressin or ANG II antagonist (20, 27) decreases baseline BP. Moreover, plasma norepinephrine but not renin activity or vasopressin was elevated after intracerebroventricular hypertonic saline for 7 days (20). In Sprague-Dawley rats (2), an acute pressor response was elicited only by intracerebroventricular injection of hypertonic saline but not by intracerebroventricular sucrose, urea, or ammonium chloride. Therefore, central loading of hypertonic saline appears to increase CSF sodium, activate sodium receptors, and cause sympathoexcitation and an increase in BP. Although intracerebroventricular hypertonic saline may increase CSF osmotic pressure, osmoreceptor-mediated increases in plasma vasopressin or plasma ANG II appear unlikely to play a significant role in this model of hypertension.

In the present study central sympathoexcitatory pathways were assessed in conscious rats by air stress and sympathoinhibitory pathways by intracerebroventricular administration of the alpha 2-adrenoceptor agonist guanabenz. Compared with rats with intracerebroventricular aCSF, in rats with intracerebroventricular sodium-rich aCSF excitatory responses to air stress and inhibitory responses to intracerebroventricular guanabenz are enhanced, indicating that the chronic sodium loading induced hypertension is associated with sympathetic hyperreactivity. An enhanced inhibitory response to intracerebroventricular guanabenz may indicate a decreased receptor occupancy or an upregulation of the alpha 2-adrenoceptors in the anterior hypothalamic area, resulting from decreased norepinephrine release to sympathoinhibitory neurons (36). Consistent with this interpretation, chronic central sodium loading reduces hypothalamic sympathoinhibition as assessed by graded electrical stimulation of the anterior hypothalamus (26). Moreover, in conscious rats intravenous infusion of hypertonic saline (2.7%) for 20 min decreased norepinephrine release in the anterior hypothalamic area (31).

Impairment of Baroreflexes

The present study shows that arterial baroreflex control of both RSNA and HR are impaired in rats intracerebroventricularly infused with 0.8 M NaCl. After chronic central sodium loading for 11 days, in anesthetized Sprague-Dawley rats, arterial baroreflex control of HR but not reflex control of RSNA was found impaired (3). These different results are likely due to the use of general anesthesia (alpha -chloralose, Ref. 3) in contrast to the use of conscious rats in the present study. General anesthesia may significantly change central baroreflex control (7).

Cardiopulmonary baroreflex control of RSNA and HR was also found impaired in centrally sodium-loaded rats. Because the volume expansion caused only minor changes (<3 mmHg) of MAP at the end of the higher rate of infusion, the impaired arterial baroreflex unlikely caused an "apparent" desensitization of the cardiopulmonary baroreflex. However, chronic effects of the decrease in gain of the arterial baroreflex on the vasomotor center and the cardiopulmonary reflex (25) cannot be excluded.

The possibility that the changes in baroreflex function are the results of increased BP (5) was not examined in the present study. However, impairment of the arterial baroreflex precedes the development of hypertension during chronic central infusion of sodium in normotensive rats (3).

Brain Sodium, "Ouabain," and ANG II

Endogenous substances with ouabain-like activity ("ouabain") in brain tissue have been well documented (22, 23, 34). Brain "ouabain" increases in rat models of salt-sensitive hypertension (22, 23), as well as in congestive heart failure, i.e., rats postmyocardial infarction or cardiomyopathic hamsters (24). In conscious rats acute intracerebroventricular hypertonic saline caused pressor effects associated with a significant increase in plasma "ouabain" and decrease in hypothalamic "ouabain" (32). In the present study chronic intracerebroventricular hypertonic saline caused sodium dose-related increases in "ouabain" in the hypothalamus and pituitary. Therefore, it appears that increased CSF sodium increases release and thereby lowers tissue content of hypothalamus "ouabain" in the acute phase, and increases release and thereafter synthesis of brain "ouabain" during chronic increases in CSF sodium.

The Fab fragments used in the present study bind ouabain and related steroids, including human and rat "ouabain" (1, 4, 24) with affinity higher than that for glycoside binding to the ATPase (4). Thus they are capable of preventing as well as reversing the actions of brain "ouabain" and ouabain on the enzyme both in vitro (24) and in vivo (12, 13, 18). So far, no aspecific effects of these Fab fragments have been noted. Previously we showed that intracerebroventricular pretreatment with these Fab fragments or losartan prevents the sympathoexcitatory and pressor responses to acute intracerebroventricular hypertonic saline in Wistar rats (17). The present study shows that in Wistar rats enhanced sympathoexcitation, impairment of baroreflexes, and hypertension by chronic central sodium loading are all prevented by concomitant intracerebroventricular Fab fragments or losartan. These findings indicate that central pathways involving both "ouabain" and ANG II also mediate the effects of chronic central sodium loading.

After blockade of the effects of vasopressin, acute intracerebroventricular hypertonic saline, ANG II, ouabain, and "ouabain" extracted from rat brain increase BP, HR, and RSNA in a similar pattern (12, 17). These responses are all abolished when the rats are pretreated intracerebroventricularly with losartan (17). In contrast, intracerebroventricular pretreatment with Fab fragments blocks responses to hypertonic saline and ouabain but not to ANG II (17). In SHR on high sodium intake (19) chronic intracerebroventricular Fab fragments caused enhanced responses to intracerebroventricular ANG II, consistent with decreased occupancy and upregulation of brain ANG II receptors presumably by blockade of brain "ouabain" causing a decrease in activity of the brain renin-angiotensin system. Thus brain ANG II receptor stimulation appears to be down-stream of brain "ouabain" in the pathways mediating the effects of central sodium. Taken together, the present study as well as the previous observations suggest the following sequence: increased CSF sodium right-arrow increased brain "ouabain" right-arrow activation of brain renin angiotensin system right-arrow sympathoexcitation, impairment of baroreflexes (10), and hypertension. However, so far no studies have assessed whether intracerebroventricular sodium indeed increases the activity of the brain renin-angiotensin system by measuring, e.g., brain renin or ANG II.

Relevance of Central Sodium For Dietary Sodium-Induced Hypertension

Brain "ouabain" and ANG II mediate the sympathetic hyperactivity (13, 18), impairment of baroreflex function (15, 16), and the exacerbation of the hypertension in rat models of salt-sensitive hypertension. However, at present it is not clear what triggers the increase in brain "ouabain." All previously mentioned effects of high sodium intake in salt-sensitive rats could be reproduced in normotensive Wistar rats with intracerebroventricular hypertonic saline, and both brain "ouabain" and ANG II mediate these effects. The present results are consistent with the concept that if dietary sodium indeed increases CSF sodium, the latter can contribute to sympathoexcitation and hypertension. Nakamura and Cowley (29) reported that 3 days after the initiation of high (4%) dietary sodium, CSF sodium rose by 4 mM/l and remained increased in Dahl S but not in Dahl salt-resistant (Dahl R) or Sprague-Dawley rats. Because the BP started to rise before the increase in CSF sodium in Dahl S rats on high sodium (29), the increase in central sodium may not be the stimulus for the initial rise of BP but could possibly contribute to the subsequent increase in BP. On the other hand, the CSF was withdrawn continuously for 24-h periods and collected between 8 and 11 AM. However, in rats the food consumption happens mainly at night, which may lead to higher CSF sodium levels during the night vs. the day. Thus, for the first 1-2 days on high sodium diet, CSF sodium levels at night may be underestimated by sampling over 24-h periods. Mozaffari et al. (28) showed that CSF sodium increased (3-4 mM/l) only transiently on day 1 after high sodium in both SHR and Wistar-Kyoto rats (WKY). Unlike the study in Dahl rats (29), Mozaffari et al. (28) collected CSF samples only acutely in anesthetized rats at an unstated time during the day, which may further underestimate the actual changes of CSF sodium. In addition, acute intracerebroventricular hypertonic saline elicited approximately threefold greater rises in BP in Dahl S vs. R (9) and in SHR vs. WKY rats (35). Therefore, in rat models of salt-sensitive hypertension, high sodium intake may result in sympathoexcitation and hypertension by causing larger increases in CSF sodium (e.g., Dahl S vs. R rats, Ref. 29) and thereafter brain "ouabain," or by causing similar increases in CSF sodium (e.g., SHR vs. WKY, Ref. 28) but larger increases in brain "ouabain" and larger sympathoexcitatory and pressor effects, or by both mechanisms. However, more specific assessments of changes in CSF sodium concentration during the feeding cycle of the rat are required to substantiate the actual patterns of changes in CSF sodium concentration by high sodium intake in salt-sensitive vs. -resistant rats.

In summary, the present study shows that the hypertension caused by chronic intracerebroventricular hypertonic saline is associated with NaCl dose-related increases in "ouabain" in hypothalamus and pituitary, increased sympathoexcitatory responses to air stress, and sympathoinhibitory responses to intracerebroventricular guanabenz, and impairment of arterial and cardiopulmonary baroreflex control of RSNA and HR. These sympathoexcitatory and pressor effects and impairment of baroreflexes are prevented by either blocking brain "ouabain" by Fab fragments or brain ANG II by losartan. To the extent that (intermittent) increases in CSF sodium by high dietary sodium occur, such increases can contribute to sympathoexcitation and hypertension by central pathways involving both brain "ouabain" and ANG II.

    ACKNOWLEDGEMENTS

Digibind was a generous gift from Glaxo Wellcome (Toronto, Canada) and losartan from DuPont Pharmaceuticals (Wilmington, DE).

    FOOTNOTES

This study was supported by operating Grant MT-11897 from the Medical Research Council of Canada and an unrestricted grant from Apotex (Toronto, Canada). S. J. Veerasingham was supported by a traineeship of the Heart and Stroke Foundation of Canada, and F. H. H. Leenen is a career investigator of the Heart and Stroke Foundation of Ontario (Ontario, Canada).

Address for reprint requests: F. H. H. Leenen, Hypertension Unit H360, Div. of Cardiology, Univ. of Ottawa Heart Institute, 1053 Carling Ave., Ottawa, Ontario, Canada K1Y 4E9.

Received 14 October 1997; accepted in final form 8 December 1997.

    REFERENCES
Top
Abstract
Introduction
Methods
Results
Discussion
References

1.  Balzan, S., U. Montali, P. Biver, and S. Ghione. Digoxin-binding antibodies reverse the effect of endogenous digitalis-like compounds on Na, K-ATPase in erythrocytes. J. Hypertens. 9, Suppl. 6: S304-S305, 1991.

2.   Bunag, R. D., and E. Miyajima. Sympathetic hyperactivity elevates blood pressure during acute cerebroventricular infusion of hypertonic salt in rats. J. Cardiovasc. Pharmacol. 6: 844-851, 1984[Medline].

3.   Bunag, R. D., and E. Miyajima. Baroreflex impairment precedes hypertension during chronic cerebroventricular infusion of hypertonic sodium chloride in rats. J. Clin. Invest. 74: 2065-2073, 1984.

4.   Butler, V. P., Jr., T. W. Smith, D. H. Schmidt, and E. Haber. Immunological reversal of the effects of digoxin. Federation Proc. 36: 2235-2241, 1977[Medline].

5.  Chapleau, M. W., G. Hajduczok, and F. M. Abboud. Peripheral and central mechanisms of baroreflex resetting. Clin. Exp. Pharmacol. Physiol. 15, Suppl.: 31-43, 1989.

6.   Davson, H., and M. B. Segal. The secretion of cerebrospinal fluid. In: Physiology of the Cerebrospinal Fluid and Blood Brain Barriers. Boca Raton, FL: CRC, 1996, p. 194-255.

7.   Fluckiger, J.-P., M. Sonnay, N. Boillat, and J. Atkinson. Attenuation of the baroreceptor reflex by general anesthetic agents in the normotensive rat. Eur. J. Pharmacol. 109: 105-109, 1985[Medline].

8.   Glowinski, J., and L. L. Iversen. Regional studies of catecholamines in the rat brain. I. The disposition of [3H]norepinephrine, [3H]dopamine and [3H]DOPA in various regions of the brain. J. Neurochem. 13: 655-669, 1966[Medline].

9.  Goto, A., L. Tobian, and J. Iwai. Potassium feeding reduces hyperactive central nervous system pressor responses in Dahl salt-sensitive rats. Hypertension 3, Suppl. I: I128-I134, 1981.

10.   Hayashi, J., K. Takeda, S. Kawasaki, Y. Nakamura, M. Oguro, T. Nakata, S. Tanabe, L.-C. Lee, S. Sasaki, and M. Nakagawa. Central attenuation of baroreflex by angiotensin II in normotensive and spontaneously hypertensive rats. Am. J. Hypertens. 1: 15S-22S, 1988[Medline].

11.   Head, G. A., and R. McCarty. Vagal and sympathetic components of the heart rate range and gain of the baroreceptor-heart rate reflex in conscious rats. J. Auton. Nerv. Syst. 21: 203-213, 1987[Medline].

12.   Huang, B. S., E. Harmsen, H. Yu, and F. H. H. Leenen. Brain ouabain-like activity and the sympathoexcitatory and pressor effects of central sodium in rats. Circ. Res. 71: 1059-1066, 1992[Abstract/Free Full Text].

13.   Huang, B. S., and F. H. H. Leenen. Brain "ouabain" mediates the sympatho-excitatory and hypertensive effects of high sodium intake in Dahl salt-sensitive rats. Circ. Res. 74: 586-595, 1994[Abstract/Free Full Text].

14.   Huang, B. S., and F. H. H. Leenen. Dietary Na and baroreflex modulation of blood pressure and RSNA in normotensive vs. spontaneously hypertensive rats. Am. J. Physiol. 266 (Heart Circ. Physiol. 35): H496-H502, 1994[Abstract/Free Full Text].

15.   Huang, B. S., and F. H. H. Leenen. Brain "ouabain," sodium, and arterial baroreflex in spontaneously hypertensive rats. Hypertension 25: 814-817, 1995[Abstract/Free Full Text].

16.   Huang, B. S., and F. H. H. Leenen. Brain "ouabain" and desensitization of arterial baroreflex by high sodium in Dahl salt-sensitive rats. Hypertension 25: 372-376, 1995[Abstract/Free Full Text].

17.   Huang, B. S., and F. H. H. Leenen. Sympathoexcitatory and pressor responses to increased brain sodium and ouabain are mediated via brain ANG II. Am. J. Physiol. 270 (Heart Circ. Physiol. 39): H275-H280, 1996[Abstract/Free Full Text].

18.   Huang, B. S., and F. H. H. Leenen. Blockade of brain "ouabain" prevents sympathoexcitatory and pressor responses to high sodium in spontaneously hypertensive rats. Am. J. Physiol. 271 (Heart Circ. Physiol. 40): H103-H108, 1996[Abstract/Free Full Text].

19.   Huang, B. S., and F. H. H. Leenen. Brain "ouabain" and angiotensin II in salt-sensitive hypertension in spontaneously hypertensive rats. Hypertension 28: 1005-1012, 1996[Abstract/Free Full Text].

20.   Kawano, Y., R. T. Sudo, and C. M. Ferrario. Effects of chronic intraventricular sodium on blood pressure and fluid balance. Hypertension 17: 28-35, 1991[Abstract/Free Full Text].

21.   Kawano, Y., K. Yoshida, H. Matsuoka, and O. Teruo. Chronic effects of central and systemic administration of losartan on blood pressure and baroreceptor reflex in spontaneously hypertensive rats. Am. J. Hypertens. 7: 536-542, 1994[Medline].

22.   Leenen, F. H. H., E. Harmsen, and H. Yu. Dietary sodium and central vs. peripheral ouabain-like activity in Dahl salt-sensitive vs. salt-resistant rats. Am. J. Physiol. 267 (Heart Circ. Physiol. 36): H1916-H1920, 1994[Abstract/Free Full Text].

23.   Leenen, F. H. H., E. Harmsen, H. Yu, and C. Ou. Effects of dietary sodium on central and peripheral ouabain-like activity in spontaneously hypertensive rats. Am. J. Physiol. 264 (Heart Circ. Physiol. 33): H2051-H2055, 1993[Abstract/Free Full Text].

24.   Leenen, F. H. H., B. S. Huang, H. Yu, and B. Yuan. Brain "ouabain" mediates sympathetic hyperactivity in congestive heart failure. Circ. Res. 77: 993-1000, 1995[Abstract/Free Full Text].

25.   Mancia, G., J. T. Shephert, and D. E. Donald. Interplay among carotid sinus, cardiopulmonary, and carotid body reflexes in the dog. Am. J. Physiol. 230: 19-24, 1976.

26.   Miyajima, E., and R. D. Bunag. Chronic cerebroventricular infusion of hypertonic sodium chloride in rats reduces hypothalamic sympatho-inhibition and elevates blood pressure. Circ. Res. 54: 566-575, 1984[Abstract/Free Full Text].

27.   Miyajima, E., and R. D. Bunag. Enhanced sympathetic pressor responses to intracerebroventricularly infused saline in awake salt-loaded rats. Am. J. Hypertens. 3: 117-122, 1990[Medline].

28.   Mozaffari, M. S., S. Jirakulsomchok, S. Oparil, and J. M. Wyss. Changes in cerebrospinal fluid Na+ do not underlie hypertensive responses to dietary NaCl in spontaneously hypertensive rats. Brain Res. 506: 149-152, 1990[Medline].

29.   Nakamura, K., and A. W. Cowley, Jr. Sequential changes of cerebrospinal fluid sodium during the development of hypertension in Dahl rats. Hypertension 13: 243-249, 1989[Abstract/Free Full Text].

30.   Oparil, S., Y.-F. Chen, Q. Meng, R. H. Yang, and J. M. Wyss. The neural basis of salt sensitivity in the rats: altered hypothalamic function. Am. J. Med. Sci. 295: 360-362, 1988[Medline].

31.   Peng, N., Q. C. Meng, S. Oparil, and J. M. Wyss. Acute saline infusion decreases norepinephrine release in the anterior hypothalamic area. Hypertension 27: 578-583, 1996[Abstract/Free Full Text].

32.   Takahashi, H., M. Matsusawa, K. Suga, I. Ikegaki, M. Nishimura, M. Yoshimura, N. Ihara, H. Yamada, and Y. Sano. Hypothalamic digitalis-like substance is released with sodium-loading in rats. Am. J. Hypertens. 1: 146-151, 1988[Medline].

33.   Takeshita, A. A., A. L. Mark, and M. J. Brody. Prevention of salt-induced hypertension in the Dahl strain by 6-hydroxydopamine. Am. J. Physiol. 236 (Heart Circ. Physiol. 5): H48-H52, 1979.

34.   Tymiak, A. A., J. A. Norman, M. Bulgar, G. C. DiDonato, H. Lee, W. L. Parker, L.-C. Lo, N. Berova, K. Makanishi, E. Haber, and G. T. Haupert. Physicochemical characterization of a ouabain isomer isolated from bovine hypothalamus. Proc. Natl. Acad. Sci. USA 90: 8189-8193, 1993[Abstract/Free Full Text].

35.   Wei, E. T., and Y. Wu. Pressor effects of intracisternal Na+ in normotensive and spontaneously hypertensive rats. Brain Res. 169: 605-609, 1979[Medline].

36.   Wyss, J. M., R. H. Yang, H. Jin, and S. Oparil. Hypothalamic microinjection of alpha2-adrenoceptor agonist causes greater sympatho-inhibition in spontaneously hypertensive rats on high NaCl diets. J. Hypertens. 6: 805-813, 1988[Medline].

AJP Heart Circ Physiol 274(4):H1269-H1276
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