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Increases in CSF [Na⁺] precede the increases in blood pressure in Dahl S rats and SHR on a high-salt diet

¹University of Ottawa Heart Institute, Ottawa, Ontario K1Y 4W7; and ²Memorial University of Newfoundland, St. John's, Newfoundland, Canada A1B 3V6

Submitted 12 February 2004 ; accepted in final form 3 May 2004

	ABSTRACT

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In Dahl salt-sensitive (S) and salt-resistant (R) rats, and spontaneously hypertensive rats (SHR) and Wistar-Kyoto (WKY) rats, at 5–6 wk of age, a cannula was placed in the cisterna magna, and cerebrospinal fluid (CSF) was withdrawn continuously at 75 µl/12 h. CSF was collected as day- and nighttime samples from rats on a regular salt intake (0.6% Na⁺; R-Na) and then on a high salt intake (8% Na⁺; H-Na). In separate groups of rats, the abdominal aorta was cannulated and blood pressure (BP) and heart rate (HR) measured at 10 AM and 10 PM, with rats first on R-Na and then on H-Na. On H-Na, CSF [Na⁺] started to increase in the daytime of day 2 in Dahl S rats and of day 3 in SHR. BP and HR did not rise until day 3 in Dahl S rats and day 4 in SHR. In Dahl R and WKY rats, high salt did not change CSF [Na⁺], BP, or HR. In a third set of Dahl S rats, sampling of both CSF and BP was performed in each individual rat. Again, significant increases in CSF [Na⁺] were observed 1–2 days earlier than the increases in BP and HR. In a fourth set of Dahl S rats, BP and HR were recorded continuously by means of radiotelemetry for 5 days on R-Na and 8 days on H-Na. On H-Na, BP (but not HR) increased first in the nighttime of day 2. In another set of Dahl S rats, intracerebroventricular infusion of antibody Fab fragments binding ouabain-like compounds (OLC) with high affinity prevented the increase in BP and HR by H-Na but further increased CSF [Na⁺]. Finally, in Wistar rats on H-Na, intracerebroventricular infusion of ouabain increased BP and HR but decreased CSF [Na⁺]. Thus, in both Dahl S and SHR on H-Na, increases in CSF [Na⁺] preceded the increases in BP and HR, consistent with a primary role of increased CSF [Na⁺] in the salt-induced hypertension. An increase in brain OLC in response to the initial increase in CSF [Na⁺] appears to attenuate further increases in CSF [Na⁺] but at the "expense" of sympathoexcitation and hypertension.

cerebrospinal fluid withdrawal; cisterna magna; Na⁺-K⁺-ATPase isoforms; ouabain-like compounds; telemetry

Why high salt intake would increase CSF [Na⁺] only in Dahl S but not in Dahl R rats (31) has not yet been clarified. In rats (31) and other species (24, 36), [Na⁺] is higher in the CSF than in plasma. Na⁺ transport across the blood-brain barrier (BBB) from the plasma to the CSF may depend on 1) epithelial sodium channels (ENaC) at the inner surface of the endothelial membrane of brain capillaries (39), including those at the choroid plexus, and probably at the epithelium covering the choroid plexus; and 2) Na⁺-K⁺-ATPase at the outer surface of the brain capillaries (1) and at the CSF side of the choroidal epithelium (22). On high salt intake, hyperactivity of ENaC and Na⁺-K⁺-ATPase may increase Na⁺ transport from the plasma to the CSF and brain interstitium. High salt intake-induced increases in brain and CSF OLC (26, 28) may then prevent a further increase in CSF [Na⁺] by inhibiting Na⁺-K⁺-ATPase on the endothelium of the brain capillaries and/or the choroidal epithelium.

In the present study, we further investigated the relation between increases in CSF [Na⁺] and in BP in Dahl S rats and in SHR when placed on high salt intake. To this end, CSF was continuously withdrawn from the cisterna magna, and CSF samples were collected over 12-h periods (daytime vs. nighttime). To verify the time course of changes in BP and heart rate (HR) on high salt intake, the abdominal aorta was cannulated for twice daily measurement of BP and HR in SHR versus WKY rats and Dahl S versus R rats and continuously by radiotelemetry in Dahl S rats. In addition, we examined whether brain OLC indeed contribute to the regulation of CSF [Na⁺]. If so, in Dahl S rats blockade of brain OLC by intracerebroventricular infusion of Fab fragments, which bind OLC with high affinity (27), will lead to a greater rise in CSF [Na⁺] on high salt intake, whereas central infusion of ouabain would increase BP but decrease CSF [Na⁺].

	METHODS

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Male, 5- to 6-wk-old Dahl S and R rats (Harlan Sprague Dawley; Indianapolis, IN), SHR, WKY rats (Taconic Farms; Germantown, NY), and Wistar rats (Charles River Breeding Laboratories, Montreal, Ontario, Canada) were housed on a 12:12-h light-dark cycle (light on from 6 AM to 6 PM and light off from 6 PM to 6 AM at the University of Ottawa; light on from 9 AM to 9 PM and light off from 9 PM to 9 AM at Memorial University) and fed a standard commercial rat chow and water ad libitum. The studies were carried out in accordance with the guidelines of the Animal Care Committees of the University of Ottawa and Memorial University of Newfoundland. Two protocols were employed. In the first protocol, the temporal relation of changes in CSF [Na⁺], BP, and HR was evaluated in rats on a high-salt diet. In the second protocol, the contribution of brain OLC to changes in CSF [Na⁺] by high-salt diet was examined. Plasma and CSF [Na⁺] and [K⁺] were measured by ion-selective electrodes on a LX20 PRO analyzer (Beckman; Coulter, CA).

Protocol 1

Experiment 1a. After 5–7 days of acclimatization in a rat sampling/infusion cage (AH 61–0042, Harvard Apparatus), under halothane inhalation anesthesia, a 23-gauge stainless steel cannula was permanently implanted into the cisterna magna of Dahl S and R rats, SHR, and WKY rats (5–7 rats for each), as described by Lai et al. (25). The cannula was secured on the skull and connected to a polyethylene tubing, which was threaded through a flexible spring and connected to a swivel (AH-61, Harvard Apparatus). The rat usually recovered 15 min after the surgery and was then placed back into the cage with the swivel connected to a low-speed, eight-channel withdrawal pump (EDCO Scientific; Chapel Hill, NC). The continuous withdrawal of CSF started in the evening at a rate of 75 µl/12 h. The CSF samples were collected over 12-h periods twice a day. Because the dead space of the cannula is 14 µl, CSF samples were collected at 8 AM and 8 PM daily (2 h later than the end of the dark and light phase), ensuring that fluid in the dead space was included in the corresponding phases. The volume of withdrawn CSF represents 3% of daily production of CSF in rats (5). Regular rat chow (0.6% Na⁺; R-Na) was provided for 3 days after the surgery and then changed to rat chow containing 8% Na⁺ (H-Na) for 6 days. Tap water was available ad libitum. The entire experiment was monitored carefully by the in-house veterinarians, and animals with any indications of infection (10%) were excluded from the study.

Experiment 1b. This experiment was performed to obtain BP and HR measurements at 10:00 AM and 10:00 PM. Dahl S and R rats, SHR, and WKY rats (5–7 rats for each) were trained with Lycra/spandex rat jackets in the sampling/infusion cage for 1 wk. Under halothane anesthesia, polyethylene tubing was then inserted into the abdominal aorta via the right femoral artery and tunneled subcutaneously to the back of the neck. The catheter was filled with heparinized saline and threaded through a flexible spring, which was secured on a rat jacket and connected to a swivel. The rats were provided with R-Na and H-Na, as described for experiment 1a. Resting BP and HR were recorded through a Grass polygraph (model 7E) for 15 min at 10 AM (daytime value) and 10 PM (evening value). Regular room light was turned on during the BP measurement in the evening. For measurement of plasma electrolytes, after the BP measurement, a 0.3-ml blood sample was withdrawn at 10 AM on day 1 with rats on R-Na and on days 2 and 5 with rats on H-Na. The blood withdrawn was replaced by 0.5 ml saline via the arterial catheter.

Experiment 2. In Dahl S rats (7–8 wk old, n = 5), the cisterna magna and a femoral artery were cannulated under halothane anesthesia in the same surgical session in each individual rat. CSF [Na⁺], BP, and HR were measured for 7 days (with rats on R-Na for 2 days and with rats on H-Na for 5 days). During the evening measurement of BP and HR, the room light was turned on only to a minimal level (red light) to maintain the dark environment. A 0.3-ml blood sample was withdrawn at 10 AM after the BP measurement on day 2 with rats on R-Na and on day 5 with rats on H-Na.

Experiment 3. In 6-wk-old Dahl S rats (n = 8), the time course of high-salt-induced changes in BP and HR was examined using radiotelemetry. As described previously (37), blood pressure telemeters (DSI model TA11PA-C40) were implanted under ketamine-xylazine anesthesia (90 and 10 mg/kg, respectively). Telemeters were positioned intraabdominally with their main body secured to the ventral abdominal muscle and their catheters inserted into the lower abdominal aorta. Penicillin G procaine suspension (30,000 units im) was administered postoperatively. The telemetry signal was processed using an analog adapter and computerized data-acquisition system, which was set to calculate and store the mean values of BP and HR over a 3-s interval every 30 s, providing 2,880 samples/day (37). Continuous recordings of BP were initiated 3 days after telemeter implantation. After being recorded for a 5-day control period on R-Na, rats were switched to H-Na and recording was continued for an additional 8 days. Telemeters offsets were measured before implantation and after removal, and the average of the pre- and postimplantation offset values was used to correct data (38).

Protocol 2

Under halothane anesthesia, a 23-gauge stainless steel cannula was implanted into the left lateral ventricle (20) in Dahl S and R rats and connected to an osmotic minipump (model 2002, Alzet) for a 14-day chronic intracerebroventricular infusion of antibody Fab fragments (Fab, Digibind, Glaxo Wellcome) or -globulins (Sigma Chemical) as a control (n = 5–8, 200 µg in 12 µl aCSF/day for both). Digibind prevents/reverses the effects of ouabain and OLC both in vivo and in vitro (23, 38) but not the effects of, e.g., angiotensin II or carbachol (17). In a group of Wistar rats, an intracerebroventricular cannula and a minipump were implanted for infusion of either ouabain (10 µg/day, n = 11, Sigma Chemical) or aCSF (n = 8) for 7–8 days. After the operation, the Dahl and Wistar rats were given a H-Na diet. At the end of the infusions, a femoral artery was cannulated under halothane inhalation in the morning, and resting BP and HR were recorded for 15 min in conscious rats in the afternoon. The rats were then anesthetized with halothane, and 100 µl CSF was collected from the cisterna magna.

Data Analyses

For the BP, HR, and CSF [Na⁺] and [K⁺] obtained in experiments 1 and 2 of protocol 1, average values for the day or night phase of 3 days on regular salt intake were used as day or night phase controls. For BP and HR obtained from telemetry, 12-h averages for the dark or light phase were used for analysis. For each rat, the averages of data recorded in each phase during the 3 days on regular salt were considered as controls. For sequential changes in [Na⁺] and [K⁺] and hemodynamics, the normality and equal variance tests were passed, and a one-way repeated-measures ANOVA was performed. When the F-values were significant for a main effect, a Duncan's test was performed for multiple comparisons. For protocol 2, a one-way ANOVA was used for the study in Dahl rats. A Student's t-test was used for the study in Wistar rats in protocol 2 as well as for comparisons between Dahl S and Dahl R rats and between SHR and WKY rats in protocol 1. Statistical significance was defined as P < 0.05.

	RESULTS

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Protocol 1

Experiment 1. The results of experiments 1a (CSF [Na⁺] measurements) and 1b [mean arterial pressure (MAP) measurements] are presented together in Figs. 1 and 2 and Table 1. There were no significant differences in daily food and water intake between Dahl S and R rats or SHR and WKY rats (data not shown). Water intake increased two- to threefold on H-Na but similarly in all strains.

View larger version (26K): [in this window] [in a new window]   Fig. 1. Changes in cerebrospinal fluid (CSF) [Na+] and in resting mean arterial pressure (MAP) and heart rate [HR; in beats/min (bpm)] in 2 groups of 5- to 6-wk-old Dahl salt-sensitive (S) rats (solid circles) vs. Dahl salt-resistant (R) rats (open squares) by high salt intake. D, daytime samples; N, nighttime samples. Data are means ± SE; n = 5–7. *P < 0.05 vs. regular salt intake.

View larger version (27K): [in this window] [in a new window]   Fig. 2. Changes in CSF [Na+] and in resting MAP and HR in 2 groups of 5- to 6-wk-old spontaneously hypertensive rats (SHR; solid circles) vs. Wistar-Kyoto (WKY) rats (open squares) by high salt intake. Data are means ± SE; n = 5–7. *P < 0.05 vs. regular salt intake.

SHR and WKY rats. On the R-Na diet, CSF [Na⁺] in the two strains was similar, but resting MAP and HR were significantly higher in SHR versus WKY rats (Fig. 2). In SHR on H-Na, CSF [Na⁺] increased significantly in the daytime of day 3 and reached plateau levels in the nighttime of day 4 (154.4 ± 1.7 vs. 149.0 ± 1.3 mmol/l, P < 0.05). Resting MAP and HR increased significantly by the night of days 4 and 3, respectively. In contrast, in WKY rats on the H-Na, there were no significant changes in CSF [Na⁺] and resting MAP or HR.

There were no significant differences in CSF [K⁺] or plasma [Na⁺] and [K⁺] between Dahl S and R rats, or between SHR and WKY rats, nor were significant changes observed on the H-Na diet (Table 1).

Experiment 2. In Dahl S rats, CSF [Na⁺], BP, and HR were all measured in each rat (Fig. 3). On the H-Na diet, CSF [Na⁺] increased significantly in the daytime of day 2, whereas BP and HR did not increase significantly until the evening of day 3. Whereas on the R-Na diet daytime and nighttime CSF [Na⁺] were similar, on the H-Na diet CSF [Na⁺] for the first 3 days was significantly greater at night. In contrast, on both the R-Na and H-Na diet, resting MAP and HR were significantly higher in the evening versus daytime, by 5 mmHg and 10 beats/min on the R-Na diet and 5–10 mmHg and 10–30 beats/min on the H-Na diet. No changes of CSF [K⁺] were observed (not shown). Compared with R-Na, H-Na did not change plasma [Na⁺] and [K⁺] (147.1 ± 1.7 vs. 146.9 ± 1.8 mmol/l and 3.4 ± 0.7 vs. 3.5 ± 0.7 mmol/l, respectively, not significant).

View larger version (24K): [in this window] [in a new window]   Fig. 3. Changes in CSF [Na+] and resting MAP and HR in the same group of 7- to 8-wk-old Dahl S rats by high salt intake. Data are means ± SE; n = 5. *P < 0.05 vs. regular salt intake; **P < 0.05, night vs. day.

View larger version (26K): [in this window] [in a new window]   Fig. 4. Changes in resting MAP and HR during day/light (D) and night/dark (N) phases in Dahl S rats on a regular salt diet for 3 days followed by a high-salt diet for 8 days. Data are means ± SE; n = 8. For each rat, the MAP and HR in each phase of each day are the average of 1,440 sampled values obtained by radiotelemetry over 12 h. P < 0.05 vs. average MAP or HR in the corresponding phase during the last 3 days on regular salt. All night values are significantly larger than day values.

In Dahl S rats with an intracerebroventricular infusion of -globulins, the H-Na diet caused the expected increases in CSF [Na⁺], MAP, and HR (Fig. 5 and Tables 1 and 2). Intracerebroventricular infusion of Fab fragments in Dahl S rats on the H-Na diet prevented the rise in MAP and HR by H-Na, but, in contrast, the rise in CSF [Na⁺] was more pronounced. Intracerebroventricular Fab fragments had no effects on MAP, HR, or CSF [Na⁺] in Dahl R rats on the H-Na diet (Fig. 4). CSF [K⁺] and plasma [Na⁺] and [K⁺] were similar in these four groups of rats.

View larger version (16K): [in this window] [in a new window]   Fig. 5. Resting MAP and CSF [Na+] and [K+] in Dahl S vs. R rats on high salt intake and simultaneous intracerebroventricular infusion of antibody Fab fragments (solid bars) or -globulins (open bars) as a control for 14 days. Data are means ± SE; n = 5–8. *P < 0.05 vs. others; **P < 0.05 vs. Dahl R rats with -globulins.

	DISCUSSION

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The major findings in the present study are as follows: 1) in Dahl S rats and SHR on high salt intake, CSF [Na⁺] increases 0.5–2 days before an increase in resting BP; and 2) in Dahl S rats on high salt intake, chronic blockade of brain OLC by intracerebroventricular Fab fragments prevents the increase in BP but leads to a greater rise in CSF [Na⁺].

In Dahl S rats on high salt intake for 6 days, CSF [Na⁺] increased gradually by 6 mmol/l. The extent of the increase in CSF [Na⁺] in rats on a H-Na diet is similar to the one observed by Nakamura and Cowley (31). In Dahl S rats in the present study, the increase in CSF [Na⁺] started in the daytime of day 2 with the H-Na diet. In the Dahl S rats studied by Nakamura and Cowley (31), CSF [Na⁺] did not increase until day 3. There are two methodological differences that may explain this difference. First, rats mainly eat at night and sampling CSF over 24-h periods as performed by Nakamura and Cowley (31) may underestimate increases in CSF [Na⁺] during the initial 1–2 nights. The present study shows differences in CSF [Na⁺] between daytime and nighttime samples (Fig. 3), and, in Dahl S rats on a H-Na diet, CSF [Na⁺] actually tended to increase as early as the night of day 1 on the H-Na diet (Figs. 1 and 3). Second, Nakamura and Cowley used female rats, 10–12 wk of age, that were placed on a 4% Na⁺ diet for 1 wk and then switched to a 8% Na⁺ diet (31), whereas the present study used male rats 5–6 and 7–8 wk of age that were directly switched to a 8% Na⁺ diet .

The BP of Dahl S rats switched to a H-Na diet first increased during the daytime of day 3 when measured by the regular intra-arterial catheter and in the nighttime of day 2 when measured by telemetry. This time pattern indicates that in the Dahl S rats studied, the pressor effect of a H-Na diet did not precede the increase in CSF [Na⁺]. In other studies in 6- to 8-wk-old male Dahl S rats using telemetry, hypertension also did not develop until several days on high salt intake (14, 33). In contrast, in 20- to 25-wk-old female Dahl S rats, the BP increased by the daytime of day 2 on a solid H-Na diet and within 24 h after the salt intake was changed from 0 to 20 mmol/day using a liquid diet (12, 31). In addition to the different protocols for the H-Na diet, it is possible that peripheral mechanisms play a more important role in the rapid development of hypertension in older female versus young male Dahl S rats. For example, the activity of 11-hydroxysteroid dehydrogenase is significantly lower in kidneys of 10- versus 6-wk-old Dahl S rats (9). This enzyme confers the specificity for the mineralocorticoid receptors, the deficiency of which may cause sodium retention and hypertension (9). On the other hand, central sodium sensitivity decreases with age in rats (20, 32). Finally, differences may result from variations in genetic characteristics, considering that a genetic contamination in Dahl rats was reported at Harlan Sprague Dawley several years ago (40).

A regular intra-arterial catheter attached to a conventional pressure transducer allows recording of BP and HR for limited times per day, with each time some disturbance of the rats. On the other hand, during telemetry recordings, BP and HR are recorded continuously during day and night phases. The rats are in their home cage, and the investigator is absent. In this setting, BP values obtained are likely a better reflection of the actual values in the active and sleep phases. On regular salt intake, both methods showed circadian rhythms, somewhat more with telemetry. On high salt intake, this phase variation markedly increased as previously reported (29). Moreover, a clear difference between the two methods emerged for both BP and HR. As measured by telemetry, the BP at night clearly and persistently increased, whereas the daytime BPs showed only modest increases. In contrast, BP measured by the regular intra-arterial catheter showed clear increases in both day and night phases. HR also showed a different pattern. As measured by the intra-arterial catheter, significant increases were noted as of day 3 with rats on a H-Na diet. In contrast, HR by telemetry showed an initial decrease without a subsequent increase. This latter pattern was also reported previously in Dahl S rats and SHR on a H-Na diet (4, 6, 29). High salt intake increases BP and HR responses to stress in Dahl S rats and SHR (15, 16). This likely explains the different findings between telemetry (day recordings without stress) versus the regular intra-arterial catheter (day recordings with stress).

The transient decrease in resting HR as well as the delay of the increase in BP relative to the increase in CSF [Na⁺] may result from an increase in plasma vasopressin as a result of increased CSF [Na⁺]. Intracerebroventricular infusion of hypertonic saline increases release of vasopressin and causes a transient decrease in HR and renal sympathetic nerve activity leading to a delayed increase in BP, but after intravenous pretreatment with a vasopressin antagonist parallel increases in sympathetic activity, BP, and HR to intracerebroventricular hypertonic saline become readily apparent (3, 8).

The present study is the first showing increases in CSF [Na⁺] in SHR on a H-Na diet. The increase in CSF [Na⁺] in SHR started in the daytime of day 3 on a H-Na diet, and CSF [Na⁺] increased by 4–5 mmol/l. In anesthetized SHR and WKY rats, Mozaffari et al. (30) withdrew acutely 150 µl of CSF through the cisterna magna at an unstated time of the day during days 1, 3, 7, and 14 on high salt intake and showed that CSF [Na⁺] increased only transiently in both SHR and WKY rats on day 1. Repeated general anesthesia and head surgery as well as acute withdrawals of 150 µl CSF may have affected food and water consumption and thereby the effect of a high salt intake on CSF [Na⁺]. In SHR, BP increased in the night of day 4 in rats on a H-Na diet. Two previous studies assessed BP by telemetry. In SHR from Harlan Sprague Dawley, BP increased in the nighttime of day 3 in rats on a H-Na diet (6). In SHR from Taconic Farms (the same as the present study) 24-h average BP increased significantly from day 4 in rats on a H-Na diet (4). Thus the onset of the increase in BP varies somewhat between studies, perhaps reflecting different breeders, but in no instance precedes the time of increase in CSF [Na⁺] noted in the present study.

The increase in CSF [Na⁺] was not paralleled by an increase in plasma [Na⁺], measured at 10 AM over 5 days in rats on a H-Na diet. In contrast, plasma [Na⁺] increased from 145 to147 mmol/l in Dahl S rats when salt intake increased from 0 to 20 mmol/day using a liquid diet (12). Another study from the same group (31) showed no effect of high salt on plasma [Na⁺] measured in the morning when a solid diet was provided. In 9- to 10-wk-old SHR and WKY rats from Harlan Sprague Dawley (7), [Na⁺] of plasma samples collected every 6 h increased significantly by 3–5 mmol/l but not until day 5 in rats on a H-Na diet. These somewhat variable results may relate to timing of blood sampling relative to recent salt and water intake.

How high salt diet increases CSF [Na⁺] in Dahl S rats and SHR, but not in Dahl R and WKY rats, has not yet been elucidated. The BBB is more permeable to Na⁺ in Dahl S versus R rats before the onset of hypertension (35). Enhanced Na⁺ transport involving ENaC may not only occur in the kidneys of Dahl S versus R rats (21) but also in the brain, because salt-sensitive hypertension can be blocked by an intracerebroventricular infusion of the Na⁺ channel blocker benzamil (11) or of mineralocorticoid antagonists (10). More active ENaC on the lumen side (39) and/or Na⁺-K⁺-ATPase on the outer surface of the endothelial membrane of cerebral capillaries as well as choroidal epithelium would enhance Na⁺ transport across the BBB from the plasma into the CSF and brain interstitium (1, 34). An increase in CSF or interstitial [Na⁺] may act as a stimulus for the production/release of OLC (17) by, e.g., astrocytes (23) as a feedback to inhibit the enhanced Na⁺ transport. Indeed, in Wistar rats on a H-Na diet, chronic intracerebroventricular infusion of ouabain markedly decreased CSF [Na⁺]. On the other hand, in Dahl S (but not Dahl R) rats on a H-Na diet, intracerebroventricular infusion of Fab fragments to bind brain OLC (18, 27) and thereby uninhibit Na⁺-K⁺-ATPase further increased CSF [Na⁺]. These results support the concept that Na⁺-K⁺-ATPase contributes to increases in CSF [Na⁺] in Dahl S rats on high salt intake. The increase in brain OLC in Dahl S rats by high salt intake (26) may then have two major effects: 1) binding to Na⁺-K⁺-ATPase involved in the regulation of CSF [Na⁺], decreasing its activity, and thereby preventing further increases in CSF [Na⁺] by high salt intake; and 2) binding to Na⁺-K⁺-ATPase in hypothalamic nuclei such as the median preoptic nucleus (2), thereby activating angiotensinergic pathways leading to sympathetic hyperactivity and hypertension as shown previously (2, 16).

Limitations of the Present Study

First, the present study does not provide direct evidence that an increase in CSF [Na⁺] actually causes the hypertension in salt-sensitive rats. The time course of the increases in CSF [Na⁺] and BP on high salt intake only strengthen the concept. Further studies are needed to substantiate the hypothesis, e.g., by preventing an increase in CSF [Na⁺] in salt-sensitive rats on high salt intake. Second, intracerebroventricular Fab fragments are specific for binding of OLC, but OLC likely have multiple sites of action in the brain. One can therefore not exclude that Fab fragments and OLC/ouabain are regulating CSF [Na⁺] at sites other than Na⁺-K⁺-ATPase on the BBB and choroid plexus. Finally, the present study does not address other major issues such as the actual cells in the brain that produce OLC in response to an increase in CSF [Na⁺] or the mechanisms of release of OLC into the CSF on the one hand and specific brain nuclei on the other hand.

Perspectives

Brain and CSF OLC may primarily increase to inhibit hyperactivity of Na⁺-K⁺-ATPase on brain capillaries and choroid plexus to prevent a further increase of the CSF [Na⁺] on high salt intake. An increase in CSF [Na⁺] by an intracerebroventricular infusion of Na⁺-rich aCSF also increases brain OLC leading to sympathoexcitation and hypertension (19), and these responses are enhanced in Dahl S versus R rats (20). In Dahl S rats and SHR, the increase in CSF [Na⁺] by high salt intake may therefore be responsible for the increase in brain OLC, sympathetic hyperactivity, and hypertension on high salt intake. In Dahl S rats, two "complementary" mechanisms, i.e., an increase in CSF [Na⁺] and enhanced responses to increased CSF [Na⁺], appear to lead to sympathetic hyperactivity and severe hypertension on high salt intake. Whether the same genetic changes are responsible for these two mechanisms and such genetic changes are the same as those involved in renal sodium homeostasis (13, 28) is an intriguing possibility.

	GRANTS

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This study was supported by Canadian Institutes of Health Research Operating Grants MOP-11897 and -6444 (to F. H. H. Leenen and B. N. Van Vliet). F. H. H. Leenen holds the Pfizer Chair in Hypertension Research, an endowed chair supported by Pfizer Canada, University of Ottawa Heart Institute Foundation, and Canadian Institutes of Health Research.

	ACKNOWLEDGMENTS

 
Digibind was a generous gift from Glaxo Wellcome, Toronto, Ontario, Canada. The authors greatly appreciate the constructive review of the manuscript by Dr. Hugh de Wardener and the technical assistance of Linda L. Chafe in telemetry experiments.

	FOOTNOTES

 

Address for reprint requests and other correspondence: F. H. H. Leenen, Hypertension Unit, Univ. of Ottawa Heart Institute, H360, 40 Ruskin St., Ottawa, Ontario, Canada K1Y 4W7 (E-mail: fleenen{at}ottawaheart.ca).

The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

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