(Hypertension. 1996;28:1005-1012.)
© 1996 American Heart Association, Inc.
Articles |
Brain "Ouabain" and Angiotensin II in Salt-Sensitive Hypertension in Spontaneously Hypertensive Rats
the Hypertension Unit, University of Ottawa (Canada) Heart Institute.
Correspondence to Frans H.H. Leenen, MD, PhD, FRCPC, Hypertension Unit, H360, Division of Cardiology, University of Ottawa Heart Institute, 1053 Carling Ave, Ottawa, Ontario, K1Y 4E9, Canada. E-mail fleenen@ohi-net.heartinst.on.ca.
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Abstract |
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Spontaneously hypertensive rats (SHR) received from 5 to 9 weeks of age a high or regular sodium diet and concomitant intracerebroventricular infusions via minipumps of the following compounds: antibody Fab fragments (200 µg/d), which bind ouabain and related steroids with high affinity; the angiotensin II (Ang II) type 1 receptor blocker losartan (1 mg/kg per day); a combination of Fab fragments and losartan; and as control,
-globulins (200 µg/d). The same doses of Fab fragments and losartan were also given intravenously. At 9 weeks of age, compared with SHR on regular sodium, SHR on high sodium that were treated with -globulins had higher resting blood pressure and showed significantly enhanced excitatory responses of blood pressure, renal sympathetic nerve activity, and heart rate to air stress and inhibitory responses to the central 
2-agonist guanabenz. Central Fab fragments and losartan alone or combined prevented all these effects of high sodium. Intravenous Fab fragments or losartan was ineffective. Compared with control SHR on high sodium, SHR on high sodium that were treated with Fab fragments had significantly increased sympathoexcitatory and pressor responses to central Ang II injection, consistent with a decrease in brain Ang II receptor occupancy. These data indicate that both increased brain "ouabain" and Ang II contribute to salt-sensitive hypertension in SHR. Brain Ang II receptor stimulation appears to be downstream of "ouabain" in the pathways mediating sympathoexcitatory and pressor effects of high sodium.
Key Words: ouabain • angiotensin II • sodium chloride, dietary • hypertension, genetic • immunoglobulins, Fab • losartan
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Introduction |
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In Dahl S rats1 and SHR,2 high dietary sodium causes sympathetic hyperactivity and increases resting BP. These effects of high sodium can be prevented by blockade of brain "ouabain"3 4 with antibody Fab fragments, which bind ouabain and related steroids5 as well as endogenous ouabain-like compound(s) ("ouabain") with high affinity.6 7 In Dahl S rats and SHR versus Dahl R rats and WKY,7 8 brain "ouabain" content is higher, and high dietary sodium further increases brain "ouabain." On the other hand, in Dahl S rats and SHR, high dietary sodium decreases the sympathoexcitatory and pressor responses to acute ICV ouabain,3 4 consistent with a downregulation or increased occupancy of brain "ouabain" receptors caused by increased brain "ouabain." We therefore proposed that brain "ouabain" mediates the sympathoexcitatory and pressor effects of high sodium in Dahl S rats and SHR.3 4
The effects of high sodium also appear to be related to increased activity of the brain RAS. The hypotensive action of ICV injection of the angiotensin-converting enzyme inhibitor captopril was greater in SHR on high versus regular dietary sodium.9 Acute microinjection of the AT1 receptor blocker losartan into the anterior hypothalamus caused a significantly greater drop of BP in SHR on high versus regular sodium intake.10 To what extent chronic blockade of the brain RAS in SHR prevents the sympathoexcitatory and hypertensive effects of high dietary sodium intake has not yet been studied. It is also unknown whether brain "ouabain" and the brain RAS contribute to salt-sensitive hypertension as two parallel systems or whether one is downstream of the other, that is, one activates the other.
An acute increase in brain sodium causes sympathoexcitatory and pressor responses.11 12 13 Both blockade of brain "ouabain" with the above-mentioned antibody Fab fragments13 14 and blockade of brain Ang II receptors by losartan14 15 or saralasin16 blunt the sympathoexcitatory and pressor responses to ICV hypertonic saline. Moreover, losartan blunts the responses to both ICV ouabain and Ang II, but Fab fragments only blunt the responses to ICV ouabain and not to ICV Ang II,14 suggesting that an increase in brain sodium first increases brain "ouabain" and that increased brain "ouabain" causes sympathoexcitation and hypertension by stimulation of the brain RAS.
In the present study, we therefore examined whether in SHR chronic blockade of brain "ouabain" and brain Ang II receptors can similarly prevent sodium-induced sympathoexcitation and hypertension and whether brain "ouabain" and the brain RAS share common pathways mediating the effects of high sodium, ie, whether the activation of the brain RAS is downstream of the activation of brain "ouabain."
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Methods |
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Male 4-week-old SHR (Taconic Farms, Germantown, NY) were housed two per cage on a 12-hour light/dark cycle and allowed a 1-week acclimatization period on normal rat chow and tap water before entering the study. At 5 weeks of age, the rats were randomly allocated to either a regular or high sodium diet (rat chow containing 120 or 1370 µmol sodium per gram of food, Harlan Sprague Dawley, Inc). The diets continued from 5 to 9 weeks of age. Tap water was freely available throughout. The study was carried out in accordance with the guidelines of the University of Ottawa Animal Care Committee.
At 5 weeks of age, with rats under sodium pentobarbital anesthesia (65 mg/kg IP), a 23-gauge stainless steel cannula was chronically implanted and fixed to the skull of the rats to serve as a guide cannula for later ICV injection, with its lower end 2.8 mm from the dura and about 0.5 mm above the right lateral ventricle.13 Coordinates were 0.4 mm posterior and 1.3 mm lateral to the bregma. In rats allocated to chronic ICV infusion, a stainless steel, right-angled cannula was implanted chronically into the left lateral ventricle using the same coordinates with a depth of 3.3 mm from the dura.3 The upper end was connected to an osmotic minipump (model 2002, Alza Corp) that was filled with either antibody Fab fragments (Digibind, Glaxo Wellcome Inc), -globulins (Sigma Chemical Co) as control (200 µg/d for both), the AT1 blocker losartan (DuPont Pharmaceuticals) (1 mg/kg per day), or Fab fragments combined with losartan at the same doses. This losartan dose was selected because in rats, ICV infusion of losartan at higher doses (10 mg/kg per day) may leak out of the central nervous system and block peripheral AT1 receptors,17 masking the possible central effects of losartan. In two rat groups, instead of ICV infusions, pumps filled with the same amounts of Fab fragments or losartan were connected to a polyethylene catheter that was permanently inserted into the right jugular vein for intravenous infusion. All compounds were dissolved in 0.9% saline. The infusion rate was 12 µL/d. The pumps were implanted subcutaneously on the back of the rats. Penicillin G (30 000 IU, Derapen, Ayerst Laboratories) was injected intramuscularly after the surgery. At 7 weeks of age, with rats under halothane anesthesia, the original pumps were replaced with new pumps filled with original compounds for ICV or intravenous infusion for another 2 weeks.
At 9 weeks of age, with rats under halothane anesthesia, polyethylene catheters were placed in the right femoral artery and vein. With additional methohexital sodium anesthesia (30 mg/kg IV, supplemented with 10 mg/kg as needed; Brevital, Eli Lilly Canada Inc), through a flank incision, a pair of platinum electrodes (A-M Systems, Inc) was placed around the left renal nerve and secured with silicone rubber (SilGel 604, Wacker).13 The catheters and electrodes were tunneled subcutaneously and externalized at the back of the neck.
Four hours after recovery from anesthesia and surgery, rats were placed in a small cage that allowed movement back and forth. The intra-arterial catheter and electrodes were connected to a Grass polygraph (model 7E), Grass 7E44 tachograph, and Grass P511 band-pass amplifier for continuous recording of BP, HR, and RSNA. The latter (spikes per second) was counted through a nerve traffic analyzer (model 706C, University of Iowa Bioengineering). Actual activity was determined by subtracting noise from total activity.13 The background noise of RSNA recording was measured by direct recording of the activity 20 minutes after the rat was killed by an overdose of sodium pentobarbital at the end of an experiment. For repeated measurements, the window setting for RSNA recording remained the same for each rat.
After a 30-minute rest followed by measurements of resting BP, HR, and RSNA, an acute environmental stress4 was provided twice at a 10-minute interval by blowing at the face of the rat for 30 seconds with a jet of air (1 to 1.5 lb/in2) from a plastic tube located 3 cm in front of the rat. The average of the responses to the two stresses was used.
Ten minutes after BP, HR, and RSNA had returned to resting levels, the 2-adrenoceptor agonist guanabenz (Sigma) dissolved in 0.9% saline was injected at doses of 25 and 75 µg ICV per 1 to 3 µL at a 5-minute interval. A microsyringe (10-µL volume, Hamilton) connected to a 26-gauge stainless steel needle was used for the injection. The needle was inserted into the ICV guide cannula so that its tip protruded into the left lateral ventricle during the injection.13
After a 20-minute rest, the arginine vasopressin antagonist [d(CH2)5Tyr(Me)]AVP (Sigma) (30 µg/kg) was injected intravenously. After 5 minutes, 30 ng Ang II (Sigma) was injected IV and ICV at a 10-minute interval. We used the arginine vasopressin antagonist to exclude the modulatory effects of vasopressin release on BP, HR, and RSNA after central Ang II injection.18 Fifteen minutes after the responses to ICV Ang II had subsided, ouabain (0.3 and 0.6 µg ICV per 1 to 2 µL) was injected at a 5-minute interval.
Subsequently, the rats on high sodium with ICV infusion pumps were anesthetized with halothane. The minipumps were removed and the rats returned to their cages. The original diets were continued, and resting BP and HR were measured at 24 and 48 hours; resting RSNA and MAP, HR, and RSNA responses to air stress were measured at 24 hours after pump removal. Rats on regular sodium treated with ICV -globulins were injected ICV with 132 µg per 4 µL -globulins, whereas rats on high sodium with intravenous Fab fragments or intravenous losartan infusion received Fab fragments (132 µg per 4 µL) or losartan (1 mg per 4 µL) ICV, respectively. Resting BP and HR were recorded at 1, 4, 18, and 24 hours and RSNA at 1 and 24 hours after these injections. Responses to air stress were also tested at 24 hours after injection. Rats rested for at least 30 minutes before each measurement of resting values, and BP, HR, and RSNA were measured for 10 minutes each time.
RSNA responses were expressed as percent changes from resting levels. Peak responses to stress and ICV injections were calculated for statistical analysis. SAS statistics system software (SAS Institute Inc) was used for analysis. Values of all variables were normally distributed, and a two-way (diet and treatment) ANOVA was performed for data between groups. When F ratios were significant, Duncan's multiple range test was used. For comparisons in the same rat before and after treatment, a paired t test was used. Statistical significance was defined at a value of P<.05.
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Results |
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Table 1
shows gains in body weight and resting MAP and HR after 4 weeks of high versus regular sodium intake. Weight gain was significantly less in SHR on high sodium treated with ICV -globulins or with intravenous Fab fragments or losartan than in SHR on regular sodium treated with -globulins. In SHR on high sodium treated with ICV Fab fragments, losartan, or both compounds, weight gain was significantly greater than in SHR on high sodium treated with -globulins but was still significantly less than that in SHR on regular sodium. Weight gain did not differ significantly among SHR on high sodium treated with ICV Fab fragments, losartan, and both compounds.
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Resting BP was significantly higher in SHR treated with -globulins on high versus regular sodium. ICV Fab fragments and losartan alone or combined fully prevented the sodium-induced increase in resting BP. Intravenous Fab fragments and losartan were ineffective. ICV losartan did not affect resting BP in SHR on regular sodium. No significant differences in resting HR were detected among all groups.
Responses to Air Stress
Air stress caused rapid increases in MAP, HR, and RSNA. Fig 1 shows that the extent of peak increases in these parameters was significantly larger in SHR on high sodium treated with -globulins than in SHR on regular sodium. This enhancement of MAP, HR, and RSNA responses by high sodium was abolished with concomitant infusion of ICV Fab fragments, losartan, or both. Intravenous administration of Fab fragments or losartan did not affect the enhanced responses. In SHR on regular sodium, ICV losartan did not change the responses to air stress.
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Responses to ICV Guanabenz
Table 2 shows that ICV guanabenz caused dose-related decreases in MAP, HR, and RSNA. The responses occurred within 1 minute of administration and lasted 5 to 15 minutes. The magnitude of peak decreases in MAP, HR, and RSNA induced by both doses was significantly larger in SHR on high sodium versus SHR on regular sodium that were treated with -globulins. In contrast, the responses to guanabenz did not differ significantly between SHR on regular sodium and SHR on high sodium that were treated with either ICV Fab fragments, losartan, or both. Intravenous administration again was ineffective. In SHR on regular sodium, ICV losartan had no effects on the responses to guanabenz.
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Responses to Ang II
After intravenous injection of the arginine vasopressin antagonist, ICV injection of 30 ng Ang II increased MAP, HR, and RSNA (Fig 2). Intravenous Ang II at this dose was ineffective (not shown). The extent of MAP, HR, and RSNA responses did not differ significantly between SHR on regular and high sodium that were treated with -globulins. Responses were significantly attenuated in SHR on regular or high sodium that were treated with ICV losartan. Compared with all other groups, SHR on high sodium that were treated with Fab fragments had responses to ICV Ang II that were enhanced two to three times. Concomitant ICV losartan blocked this enhancement. Intravenous Fab fragments or losartan had no effects on the responses to ICV Ang II.
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Responses to Ouabain
Central injection of ouabain at 0.3 (not shown) and 0.6 µg caused dose-related increases in MAP, HR, and RSNA. Fig 3 shows that the extent of MAP, HR, and RSNA responses to ouabain was significantly less in all rat groups on high sodium as well as on regular sodium that were treated with ICV losartan than the extent of responses in SHR on regular sodium that were treated with -globulins.
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Acute Withdrawal of Fab Fragments and/or Losartan
Fig 4 shows that up to 48 hours after removal of infusion pumps, resting MAP remained elevated around 170 mm Hg (Fig 4, top) in SHR on high sodium that were treated with ICV -globulins. In rats on high sodium that were treated with ICV losartan, resting MAP remained around 135 mm Hg (Fig 4, top). In SHR on high sodium that were treated with ICV Fab fragments or Fab fragments plus losartan, resting MAP increased significantly by 15 to 35 mm Hg at 24 and 48 hours after removal of the pump but was still significantly lower than values in rats on high sodium that were treated with -globulins.
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Resting RSNA decreased to 64±3% of the original activity 24 hours after removal of the pumps in SHR on high sodium that were treated with -globulins; this decrease was probably in part related to the deterioration in the quality of RSNA recording over time. In contrast, resting RSNA was 84±4%, 73±3%, and 80±3% of the original activities in SHR on high sodium that were treated with ICV Fab fragments, losartan, or both (P<.05 for all versus SHR treated with -globulins) (Fig 4
, bottom). In SHR on high sodium that were treated with -globulins, the extent of peak responses of MAP, HR, or RSNA to air stress remained enhanced similarly to that before removal of the pumps (not shown). In SHR treated with Fab fragments or Fab fragments plus losartan, responses to air stress 24 hours after removal of the pumps were enhanced compared with responses before removal (MAP: 25±3 versus 16±2 mm Hg or 30±2 versus 14±2 mm Hg; HR: 31±3 versus 20±2 beats per minute or 30±3 versus 19±3 beats per minute; RSNA: 46±4% versus 20±2% of resting or 42±2% versus 18±2% of resting; P<.05 for all). At 24 hours after removal of pumps, in SHR treated with losartan alone, MAP responses to stress were still decreased and similar to responses before removal (20±4 versus 18±2 mm Hg), whereas RSNA and HR responses were slightly but significantly enhanced compared with responses before removal (HR: 26±4 versus 18±3 beats per minute; RSNA: 30±4% versus 21±2% of resting; P<.05 for both).
Acute ICV Injection of Fab Fragments or Losartan
In SHR on high sodium that were previously treated with intravenous Fab fragments, acute ICV Fab fragments did not change resting MAP at 1 and 4 hours after injection, but MAP was significantly decreased at 18 and 24 hours (Fig 5, top). In SHR on high sodium and intravenous losartan, MAP dropped markedly at 1 hour after ICV losartan, to a lesser extent at 4 hours, and had returned to baseline at 18 and 24 hours after injection. Compared with SHR on regular sodium that were treated with -globulins, SHR on high sodium that were previously treated with intravenous Fab fragments had resting RSNA values that did not change significantly 1 hour after ICV Fab fragments (not shown) but decreased significantly at 24 hours (53±2% versus 62±3% before injection, P<.05) (Fig 5, bottom). Compared with SHR on regular sodium that were treated with -globulins, SHR on high sodium and intravenous losartan had resting RSNA values that were significantly decreased 1 hour after ICV losartan (80±3% versus 97±4% before injection, P<.05) but not significantly decreased 24 hours after ICV losartan (Fig 5, bottom). Compared with responses before injections, the extent of peak responses to air stress 24 hours after injection were not significantly changed in SHR treated with -globulins and losartan (not shown) but were significantly attenuated in SHR treated with Fab fragments (MAP: 19±3 versus 29±2 mm Hg; HR: 26±3 versus 42±4 beats per minute; RSNA: 30±6% versus 52±5% of resting; P<.05 for all).
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Discussion |
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We previously showed that in Dahl S rats and SHR, high dietary sodium increases sympathetic activity and resting arterial BP and that these sympathoexcitatory and hypertensive effects of high sodium are probably mediated by an increase in brain "ouabain."3 4 The present study shows that both brain "ouabain" and the brain RAS mediate these effects. Moreover, brain "ouabain" and brain Ang II appear to share a common pathway, and brain Ang II receptor stimulation occurs downstream of brain "ouabain" receptor stimulation.
High dietary sodium causes sympathoexcitation and exaggeration of the development of hypertension in rats genetically predisposed to hypertension, such as SHR4 and Dahl S rats.3 Whether an increase in brain sodium is responsible for the salt-sensitive hypertension is still controversial.19 20 In both SHR and WKY,19 the sodium concentration in CSF was increased after 1 day of high sodium intake, but after 3 days of high sodium, CSF sodium was increased only in WKY. In contrast, Nakamura and Cowley20 reported that high sodium intake increased CSF sodium only in Dahl S and not Dahl R rats. Unlike the study in Dahl rats,20 in SHR and WKY,19 CSF samples were taken only during the day, which probably does not reflect the changes after food intake, ie, during the evening or night. When CSF sodium did increase after high dietary sodium,19 20 the extent of increase was close to the increase induced by chronic ICV hypertonic saline,21 22 which also causes sympathoexcitation and hypertension.
In SHR and Dahl S rats, high dietary sodium increases both resting BP and sympathetic activity. In the present study, the latter was estimated by sympathoexcitatory and pressor responses to air stress and sympathoinhibitory and depressor responses to guanabenz. An enhanced inhibitory response to 2-adrenoceptor agonists such as guanabenz or clonidine generally reflects an upregulation and/or decreased 2-receptor occupancy in the anterior hypothalamus23 24 as a result of decreased norepinephrine release from sympathoinhibitory neurons. ICV administration of Fab fragments, which bind ouabain and human and rat "ouabain" with high affinity,5 6 7 prevents as well as reverses increased sympathetic activity and increased BP in Dahl S rats and SHR on high dietary sodium.3 4 Thus, brain "ouabain" appears to mediate salt-sensitive hypertension in these hypertensive rats. In the present study, intravenous Fab fragments at a dose identical to the one used for ICV infusion did not affect the sympathoexcitatory and pressor effects of high dietary sodium, confirming further that ouabain mediates salt-sensitive hypertension via central mechanisms.
An increased activity of the brain RAS has been reported in SHR on regular sodium. At 2 to 20 weeks of age, brain Ang II levels were higher in SHR versus WKY.25 Moreover, compared with WKY, SHR showed increased CSF Ang II levels,26 brain Ang II turnover,26 affinity of brain Ang II receptors,27 and brain angiotensinogen mRNA.28 Injection of losartan into the anterior hypothalamus caused a greater decrease in BP in SHR at 9 weeks of age versus age-matched WKY.29 However, in SHR at 20 weeks of age, ICV injection of losartan did not cause a significant decrease in resting BP over the next 48 hours.30 In the present study, chronic ICV losartan did not affect the development of hypertension or sympathetic activity in SHR on regular sodium. This is consistent with a study by Kawano et al17 showing that in SHR aged 12 to 16 weeks, ICV losartan for 2 weeks at the same dose as used in the present study did not affect resting BP. In the same study,17 administration of losartan at a higher dose of 10 mg/kg per day ICV or SC for 2 weeks decreased resting BP to the same extent, indicating that the depressor effect of ICV losartan at a higher dose is a result of leakage of the compound out of the central nervous system and blockade of peripheral AT1 receptors. Okuno et al31 showed that in SHR, ICV administration of the angiotensin-converting enzyme inhibitor captopril from 7 to 11 weeks of age partially (about 20 mm Hg) attenuated the development of hypertension without changing sympathetic activity. However, ICV angiotensin-converting enzyme inhibitors also may increase brain bradykinin or neuropeptide levels.32 33 Therefore, whereas peripheral blockade of the RAS is very effective in preventing the development of hypertension in SHR17 33 34 and brain RAS activity may be higher in SHR than in WKY, chronic blockade of the brain RAS by ICV losartan at the dose used appears not to be able to prevent the development of hypertension. Since an acute injection of 20 µg losartan into the anterior hypothalamic area caused depressor responses in SHR on regular sodium,29 it is possible that chronically administered ICV losartan at the present dose may not reach certain areas in the brain to prevent the development of genetic hypertension. Alternatively, in SHR on regular sodium, the RAS in these areas may play a role in the acute regulation of BP but may not be involved in the development of hypertension.
In contrast, increased activity of the brain RAS appears to play a major chronic role in salt-sensitive hypertension in SHR. Takata et al9 reported that SHR on high versus regular dietary sodium show an enhanced depressor response to ICV injection of captopril. Moreover, microinjection of losartan into the anterior hypothalamus caused larger depressor effects in SHR on high versus regular dietary sodium from 7 to 10 weeks of age.35 However, so far no studies have established whether both the chronic sympathoexcitatory and pressor responses to high sodium depend on the brain RAS. In the present study, chronic blockade of brain Ang II receptors by ICV losartan prevented both the sympathetic hyperactivity and exacerbation of hypertension in SHR on high sodium. Teruya et al36 reported recently that chronic ICV infusion of the AT1 receptor blocker CV-11974 concomitantly with high dietary sodium prevented the development of hypertension in Dahl-Iwai salt-sensitive rats. Therefore, activation of brain Ang II receptors appears to be essential for the development of salt-sensitive hypertension in both SHR and Dahl S rats.
The present study shows that ICV administration of Fab fragments, losartan alone, or Fab fragments and losartan combined prevents in a similar pattern the enhanced hypertension and enhanced responses to air stress and ICV guanabenz by high sodium intake. Blockade of either brain "ouabain" or brain Ang II receptors therefore prevents sodium-induced sympathetic hyperactivity and hypertension. The pattern of responses to ICV ouabain and Ang II suggests that rather than two parallel pathways, the two systems interact and are involved in the same pathways mediating sympathoexcitatory and pressor effects of high dietary sodium. Chronic blockade of brain Ang II receptors with losartan blunted responses to acute ICV injection of both ouabain and Ang II. Chronic blockade of brain "ouabain" did not blunt but significantly enhanced excitatory responses of BP, HR, and RSNA to acute ICV injection of Ang II, suggesting that blockade of brain "ouabain" resulted in decreased activity of the brain RAS and therefore upregulation or decreased occupancy of brain Ang II receptors. Thus, similar to our study assessing the role of brain "ouabain" and brain Ang II in the acute effects of ICV hypertonic saline,14 it appears that in response to high sodium intake and possibly increased brain sodium, brain "ouabain" activation occurs somewhere upstream of brain RAS activation and AT1 receptor stimulation. If this is the case, one would expect that high sodium alone would increase the occupancy of or would downregulate brain Ang II receptors, leading to a decrease in responses to ICV Ang II, similar to that observed for ICV ouabain (see Fig 3). In the present study, however, the responses to ICV Ang II did not differ significantly between groups on regular and high sodium that were treated with -globulins (see Fig 2). Thus, it appears that the pathways involving Ang II receptor stimulation in salt-induced hypertension in SHR are not the only pressor pathways involving AT1 receptors. Responses to ICV Ang II may involve several pathways, only some of which are affected by high sodium.
Although the time course is not identical, in SHR on high sodium intake, changes in resting BP and RSNA in response to acute injection or withdrawal of Fab fragments and losartan are in the same direction; ie, the injections cause decreases in MAP and RSNA, but withdrawal causes increases in MAP and RSNA relative to controls. ICV injection of 1 mg losartan decreased resting BP about 20 and 10 mm Hg at 1 and 4 hours, respectively, after the injection and had no depressor effects at 18 and 24 hours. These responses are similar to the findings by Yang et al35 showing that in SHR on high sodium, microinjection of 40 µg losartan into the anterior hypothalamus caused a peak depressor response of about 23 mm Hg at 70 minutes after injection. In SHR on high sodium, withdrawal of both the Fab fragments and losartan also enhanced the BP and RSNA responses to air stress, whereas ICV injection of the two compounds normalized the enhanced responses. These observations further support the hypothesis that tonically active brain "ouabain" and the brain RAS contribute to sodium-induced sympathoexcitation and hypertension in SHR.
A functional relationship of brain "ouabain" and brain Ang II is also supported by histological findings regarding ouabain containing neurons and the brain RAS. Ouabain-immunopositive neurons are distributed in rat hypothalamic regions such as paraventricular and supraoptic nuclei,37 38 and the nerve fibers are distributed abundantly in areas such as the anteroventral third ventricle including the organum vasculosum of the lamina terminalis, the subfornical organ, and the median eminence. In these areas, Ang II receptors39 and other components of the brain RAS40 are densely present. In addition, studies with brain lesions indicated that the anteroventral third ventricle is also crucial to the development of salt-sensitive hypertension41 and the pressor effects of ICV hypertonic saline,42 ouabain,43 and Ang II.44
Previous studies45 46 suggested that brain Ang II may be involved in the release of "ouabain" in the brain. This conclusion was based on the observation that acute45 or chronic46 ICV administration of Ang II increased plasma "ouabain." Circulating plasma "ouabain" can be of peripheral47 and central48 49 origins, the latter presumably released from the pituitary. Central injection of Ang II causes sympathoexcitatory and pressor effects14 50 as well as release of vasopressin,18 and in parallel, the pituitary may therefore release "ouabain" as well.
In summary, we demonstrated that increased brain "ouabain" and brain Ang II are both involved in mediating sympathoexcitatory and pressor effects of high sodium intake in SHR. Similar to our study evaluating the role of "ouabain" and Ang II in the effects of acutely increased brain sodium, high dietary sodium appears to increase brain "ouabain" first, and increased brain "ouabain" activates the brain RAS, leading to sympathoexcitation and hypertension. This concept provides significant new insights into our understanding of the mechanisms contributing to sodium-induced sympathoexcitation and hypertension and may lead to more-specific therapeutic interventions for the treatment of salt-sensitive hypertension.
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Acknowledgments |
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This work was supported by operating grants from the Medical Research Council of Canada and an unrestricted grant from Apotex Inc, Canada. F.H.H.L. is a Career Investigator of the Heart and Stroke Foundation of Ontario (Canada). Digibind and losartan were generous gifts from Glaxo Wellcome Inc, Toronto, Canada, and DuPont Pharmaceuticals, Wilmington, Del, respectively.
Received April 4, 1996; first decision June 17, 1996; accepted July 8, 1996.
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References |
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