Abstract To assess the possible role of brain “ouabain” in modulating arterial baroreflex function in salt-sensitive hypertension, arterial baroreflex control of renal sympathetic nerve activity and heart rate was evaluated in conscious spontaneously hypertensive rats and compared with that in Wistar-Kyoto rats. A regular sodium or high sodium diet was provided from 5 to 9 weeks of age, with intracerebroventricular infusion of antibody Fab fragments, which bind ouabainlike substances with high affinity, or, as control, nonspecific γ-globulins (200 μg · 12 μL−1 · d−1 for both). Baroreflex function was assessed by plotting changes in renal sympathetic nerve activity or heart rate against changes in mean arterial pressure by phenylephrine and nitroprusside. In control Wistar-Kyoto rats, high sodium intake did not increase resting blood pressure but sensitized baroreflex control of renal sympathetic nerve activity. In control spontaneously hypertensive rats, high sodium intake significantly increased blood pressure but did not enhance renal sympathetic nerve activity responses. However, in spontaneously hypertensive rats given high sodium diets and treated with Fab fragments, blood pressure did not increase and the baroreflex control of renal sympathetic nerve activity was sensitized significantly. We conclude that in spontaneously hypertensive rats, increase of central “ouabain” by high sodium intake prevents an increase in the sensitivity of arterial baroreflex control of renal sympathetic nerve activity, as observed in Wistar-Kyoto rats on high sodium diets.
In rats genetically predisposed to hypertension, sodium-sensitive hypertension may be partly related to sodium-induced changes in arterial baroreflex function. Sensitization of arterial baroreflex control of renal sympathetic nerve activity (RSNA), as observed in, for example, Wistar-Kyoto (WKY) or Dahl salt-resistant (DR) rats, may prevent sodium-induced volume expansion and therefore the development of hypertension.1 2 In normotensive rats, high sodium intake (H-Na) increases resting blood pressure only when combined with chronic sinoaortic denervation.1 3 In young spontaneously hypertensive rats (SHR), H-Na exaggerates the development of hypertension and does not sensitize the arterial baroreflex.1 H-Na exacerbates baroreflex impairment and induces hypertension in Dahl salt-sensitive (DS) rats.4 The mechanisms responsible for these different changes of baroreflex sensitivity by H-Na in SHR and DS compared with their normotensive controls are still unclear.
In recent years, evidence has increased to support the existence of endogenous compounds with ouabainlike activity (“ouabain”) both peripherally5 and in the brain.6 In normotensive rats,7 chronic intracerebroventricular infusion of hypertonic saline induces hypertension as well as impairment of the arterial baroreflex, and the latter precedes the former. In conscious rats,8 acute intracerebroventricular infusion of hypertonic saline, ouabain, or brain “ouabain” elicits similar sympathoexcitatory and pressor responses. These responses can be blocked by intracerebroventricular pretreatment with antibody Fab fragments, which bind ouabainlike substances with high affinity.8 Brain “ouabain” is increased in SHR or DS compared with WKY or DR, and H-Na further increases brain “ouabain” in SHR and DS.6 9 Recently we showed that increased central “ouabain” appears to be responsible for decreased sympathoinhibition, increased sympathoexcitation, desensitized arterial baroreflex function, and the development of hypertension in DS rats on H-Na.10 11 In the present study, we examined the possible role of brain “ouabain” in modulating the arterial baroreflex and the exaggeration of hypertension in SHR in response to H-Na.
Male SHR and WKY (Taconic Farms) were randomly allocated from 5 to 9 weeks of age to either a regular sodium diet (R-Na) or H-Na (120 and 1370 μmol sodium, respectively, per gram of food; Harlan Sprague Dawley Inc) with free access to tap water. All experimental procedures were carried out in accordance with the guidelines of the University of Ottawa Animal Care Committee for the use and care of laboratory animals. When the rats were 5 weeks of age, and while they were under sodium pentobarbital anesthesia, a stainless steel, right-angled cannula was implanted into the left brain lateral ventricle of each rat, as described previously.10 The cannula was connected to an osmotic minipump (model 2002, Alza Corp), which was filled with either antibody Fab fragments (Digibind, Burroughs Wellcome Inc) or nonspecific γ-globulins (Sigma Chemical Co). Both compounds were dissolved in saline. The infusion rate was 200 μg · 12 μL−1 · d−1. The pumps were implanted subcutaneously on the backs of the rats. When the rats were 7 weeks old, while they were under halothane anesthesia, the original minipumps were replaced with new pumps filled with the same compounds for intracerebroventricular infusion for another 2 weeks.
When the rats were 9 weeks old, while they were under halothane anesthesia, catheters were placed in the right femoral artery and jugular vein. While they were under methohexital sodium anesthesia (30 mg/kg IV supplemented with 10 mg/kg as needed; Brevital, Eli Lilly Canada Inc), a pair of platinum electrodes (A-M System Inc) was placed around the left renal nerve and secured with silicone rubber (SilGel 604, Wacker).1 The electrodes and catheters were tunneled subcutaneously to the back of the neck.
Four hours after recovery from the anesthesia, each rat was placed in a small cage without restriction of movement. The intra-arterial catheter and electrodes were connected to a Grass 7E polygraph (Grass Instrument Co), a Grass 7P44 tachograph, and a Grass P511 bandpass amplifier. RSNA (spikes per second) was counted through a nerve traffic analyzer (model 706C, University of Iowa Bioengineering). The actual RSNA was determined by subtracting noise from the total activity.1
After a 30-minute stabilization, basal mean arterial pressure (MAP), heart rate (HR), and RSNA were recorded. Phenylephrine (5 to 50 μg · kg−1 · min−1) in 5% dextrose was infused at increasing doses to induce a ramp increase of MAP by 50 mm Hg over 2 to 3 minutes. Twenty minutes after blood pressure, RSNA, and HR had returned to the basal levels, nitroprusside (5 to 100 μg · kg−1 · min−1) in 5% dextrose was infused to induce a ramp decrease of MAP by 50 mm Hg. Infusion speed for both was less than 0.08 mL/min.
The resting RSNA of each rat was considered as 100%, and responses of RSNA were expressed as percentage of resting value. The RSNA and HR responses were plotted against the changes in MAP evoked by phenylephrine and nitroprusside infusion. The changes in RSNA (ΔRSNA) or HR (ΔHR) in response to changes in MAP were analyzed as a logistic curve1 using the logistic equation
where P1 is the lower ΔRSNA plateau, which represents the maximal decrease in RSNA; P2 is the ΔRSNA range; P3 is a curvature coefficient; and P4 is ΔMAP50, ie, ΔMAP at half the ΔRSNA range. The average gain (G), or slope of the curve between the two inflection points of the curve, is given by G=−P2×P3/4.56, which represents the overall baroreflex sensitivity tested by both increasing and decreasing blood pressure. For a separate analysis of RSNA and HR responses to increases and decreases in blood pressure, the gains of responses were obtained by analyzing the linear portion of each curve by linear regression. For all comparisons, the data were assessed using ANOVA followed by the Duncan multiple range test. The level of statistical significance was set at P<.05.
Resting MAP was significantly increased by H-Na in control SHR compared with control SHR on R-Na (Table⇓). In contrast, MAP was not increased by H-Na in SHR treated with intracerebroventricular Fab fragments compared with SHR on R-Na treated with Fab fragments or control SHR on R-Na. H-Na had no effects on resting MAP in WKY. Fab fragments did not affect the resting MAP in SHR on R-Na or in WKY on either diet. There were no significant differences in resting HR among all groups of rats (data not shown).
Baroreflex Control of RSNA
Data for the baroreflex control of RSNA are shown in the Table⇑ and Figure⇓. Nitroprusside- and phenylephrine-induced ramp decreases and increases in MAP caused ramp excitatory and inhibitory responses of RSNA. In control WKY, H-Na significantly enhanced RSNA responses, as reflected by an increase in average gain of the RSNA-MAP baroreflex curve. In contrast, in control SHR, the average gain of the baroreflex curve was similar on H-Na as on R-Na, and on H-Na it was significantly lower than in WKY that were also on H-Na. However, in SHR on H-Na and treated with intracerebroventricular Fab fragments, the average gain of the RSNA response was significantly increased compared with SHR similarly treated with Fab fragments while on R-Na. Fab fragments had no effect on the baroreflex control of RSNA in SHR on R-Na or in WKY on either diet.
In concurrence with the results obtained by logistic analysis, when RSNA responses to phenylephrine- and nitroprusside-induced changes in MAP were analyzed separately, H-Na was seen to sensitize reflex control of RSNA, as tested by either phenylephrine or nitroprusside infusions, in control WKY but not in control SHR. In SHR on H-Na, treatment with Fab fragments significantly increased the gain of reflex control of RSNA as tested by either agent.
Baroreflex Control of HR
Data on the baroreflex control of HR are shown in the Table⇑. On R-Na, the average gain of HR responses to changes in MAP was significantly decreased in SHR compared with WKY. In control WKY, H-Na had no effects on average gain of HR responses to changes in MAP. In control SHR, the average gain of HR responses to changes in MAP was slightly decreased by H-Na compared with R-Na (P=.09). This decrease was prevented by treatment with Fab fragments (P<.05).
When the HR responses to phenylephrine and nitroprusside were analyzed separately, in SHR patterns of change in gains were observed similar to those obtained by analyzing the data using logistic analysis (data not shown). In control WKY, H-Na desensitized HR responses to nitroprusside-induced but not to phenylephrine-induced changes in MAP compared with responses in WKY on R-Na (nitroprusside, −1.86±0.14 compared with −2.22±0.12 beats per minute per millimeter of mercury (bpm/mm Hg), P<.05; phenylephrine, −2.84±0.17 compared with −3.16±0.21 bpm/mm Hg, P=NS). Fab fragments had no effects on the gain of HR-MAP curves in WKY on either diet.
The present study shows that, in contrast to the case in young WKY, H-Na in young SHR does not sensitize arterial baroreflex control of RSNA. However, after blockade of brain “ouabain” by antibody Fab fragments, H-Na also sensitizes baroreflex control of RSNA in SHR and no longer increases the blood pressure.
The changes in baroreflex control of RSNA by dietary sodium shown in the present study are consistent with several previous studies. In normotensive rats, H-Na sensitizes the arterial baroreflex with no effects on resting blood pressure.1 2 3 In SHR and DS rats, H-Na increases blood pressure but has no effects on baroreflex sensitivity1 2 or even causes desensitization.4 11 Because H-Na induces hypertension in normotensive rats after sinoaortic denervation,1 3 in SHR or DS rats desensitization or lack of sensitization appears to facilitate the expression of salt-sensitive hypertension.
Both peripheral and central mechanisms may be involved in the sodium-induced changes in the arterial baroreflex. In anesthetized DR but not in DS rats,2 H-Na increased baroreceptor sensitivity, as reflected by enhanced afferent aortic depressor nerve activity in response to an increase in blood pressure. However, bradycardia elicited by electrical stimulation of the aortic depressor nerve was attenuated in DS rats on H-Na,4 suggesting a central modulation of the baroreflex by sodium. In anesthetized SHR,12 H-Na attenuated RSNA and HR responses to changes in MAP as well as RSNA but not HR responses to electrical aortic depressor nerve stimulation, indicating that H-Na impaired both overall and central control of the baroreflex.
The mechanisms of sodium-induced changes in baroreflex sensitivity are still unclear. Chronic intracerebroventricular hypertonic saline causes impairment of baroreflex function and subsequently hypertension in normotensive rats.4 Acute intracerebroventricular hypertonic saline induces sympathoexcitatory and pressor effects in conscious rats,8 and these effects can be prevented by blockade of brain “ouabain” with intracerebroventricular antibody Fab fragments. Dietary sodium–induced increase in brain “ouabain” is more marked in SHR6 and DS rats9 than in their normotensive controls. In conscious DS rats, H-Na causes impairment of baroreflex control of RSNA and hypertension, and blockade of brain “ouabain” both normalizes the baroreflex and prevents the hypertension.11 On the other hand, ouabain sensitizes arterial baroreceptor function by means of peripheral mechanisms.13 We therefore proposed that in salt-sensitive substrains such as DS rats, increased brain “ouabain” may play a dominant role in the impairment of baroreflex function, and peripheral “ouabain” may play only a minimal role in the modulation, particularly when an impairment of baroreceptor function exists prior to H-Na.14 The present study provides evidence supporting this hypothesis, because in control SHR, H-Na did not sensitize the baroreflex but H-Na plus central Fab fragments did, as observed in control WKY on H-Na. Central Fab fragments block brain “ouabain” and thereby appear to prevent the impairment of the baroreflex at the central level but may not affect the increase in peripheral “ouabain” that can sensitize the baroreflex.
The present data do not exclude the possibility that sensitization of the arterial baroreflex in SHR on H-Na treated with Fab fragments is the result of prevention of more severe hypertension rather than blockade of a brain “ouabain” mechanism specifically involved in baroreflex modulation. Moreira et al15 found that in renal hypertensive rats, acute unclipping reverses hypertension and causes rapid normalization of the desensitized peripheral component of the arterial baroreflex. They suggested that the baroreflex desensitization may be secondary to the increase in resting blood pressure. However, in DS rats on H-Na, 6-hydroxydopamine prevents the increase in blood pressure, but the absence of baroreflex sensitization remains.2
Consistent with the results of our previous studies,1 a dissociation of baroreflex control of HR from that of RSNA was observed in the present study. In control WKY, H-Na sensitized reflex control of RSNA but had no effects (by logistic analysis) on or even desensitized (by linear regression) the reflex control of HR. Pretreatment with Fab fragments did not affect these responses. In contrast, in control SHR H-Na had no effects on reflex control of RSNA and tended to desensitize the reflex control of HR. In SHR on H-Na, Fab fragments sensitized reflex control of RSNA as well as prevented the tendency toward desensitization of reflex control of HR. Arterial baroreflex control of HR in conscious rats is predominantly mediated by the parasympathetic nervous system.16 Changes in the control of HR versus RSNA by H-Na therefore depend on the balance of the effects of H-Na on the sympathetic and parasympathetic nervous systems.
In conclusion, H-Na does not cause sensitization of arterial baroreflex control of RSNA in SHR, in contrast to WKY. Blockade of brain “ouabain” by antibody Fab fragments sensitizes baroreflex control of RSNA and prevents the exacerbation of hypertension in SHR on H-Na. Fab fragments had no effects on baroreflex or resting blood pressure in SHR on R-Na or in WKY on R-Na or H-Na. These results support the concept that sodium-induced increase in brain “ouabain” impairs the arterial baroreflex, thereby facilitating the exacerbation of hypertension in SHR.
This work was supported by an operating grant from the Medical Research Council of Canada. F.H.H.L. is a Career Investigator of the Heart and Stroke Foundation of Ontario.
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