Abstract In Dahl salt-sensitive (S) rats, high sodium intake further desensitizes arterial baroreflex function. To assess the possible involvement of brain “oubain,” we gave Dahl S rats a regular or high sodium diet from 4 to 7 weeks of age and administered intracerebroventricular antibody Fab fragments, which bind ouabain with high affinity, or γ-globulins as control (200 μg/12 μL per day for both) using osmotic minipumps. We assessed arterial baroreflex function by plotting changes in renal sympathetic nerve activity or heart rate against changes in mean arterial pressure of conscious rats elicited by intravenous phenylephrine and nitroprusside. Dahl S rats on high sodium treated with γ-globulins showed a significantly higher resting mean arterial pressure versus other rats (130 to 140 versus 95 to 105 mm Hg). In rats treated with γ-globulins, high sodium desensitized baroreflex control of renal sympathetic nerve activity compared with rats on regular sodium (average gain: −1.88±0.12 versus −2.73±0.13, P<.05). In contrast, in rats treated with Fab fragments, high sodium did not increase blood pressure and did not desensitize but slightly sensitized reflex control of renal sympathetic nerve activity. Changes in reflex control of heart rate were similar to those of renal sympathetic nerve activity. These data indicate that blockade of brain “oubain” prevents sodium-induced hypertension as well as the desensitization of the arterial baroreflex in Dahl S rats. Increased brain “oubain” may desensitize the arterial baroreflex and thereby facilitate the hypertension in Dahl S rats on high sodium.
Different changes in arterial baroreceptor reflex function in response to high sodium intake in genetically hypertensive rats versus normotensive controls have been proposed to be causative factors of sodium-sensitive hypertension. In Wistar-Kyoto rats (WKY), high sodium sensitizes arterial baroreflex control of renal sympathetic nerve activity (RSNA) without increasing resting blood pressure (BP)1 but increases BP in WKY with chronic sinoaortic denervation.1 2 In young spontaneously hypertensive rats (SHR), high sodium does not sensitize the arterial baroreflex and exaggerates the development of hypertension.1 The lack of sensitization in SHR may facilitate the further increase in BP.1 Similarly, in Dahl salt-resistant (Dahl R) rats, high sodium sensitizes arterial baroreflex without changing BP3 but exacerbates baroreflex impairment and induces hypertension in Dahl salt-sensitive (Dahl S) rats.4 The mechanisms responsible for these different changes of baroreflex function by high sodium in SHR and Dahl S rats versus their normotensive controls have not yet been explored.
In normotensive rats,5 long-term intracerebroventricular (ICV) infusion of hypertonic saline induces hypertension and impairs the arterial baroreflex, with the latter preceding the former. In conscious rats,6 short-term ICV infusion with hypertonic saline, ouabain, or brain tissue extracts containing ouabainlike activity (“oubain”) elicits similar sympathoexcitatory and pressor responses. These responses can be blocked by pretreatment with ICV antibody Fab fragments (Digibind),6 which bind ouabain with high affinity. Increased central “oubain” appears to mediate the sodium-sensitive hypertension in SHR6 and Dahl S rats7 on a high sodium intake. In the present study, we examined the possible role of brain “oubain” in the impairment of the arterial baroreflex in Dahl S rats in response to a high sodium intake. Brain “oubain” appears to be an essential mechanism because both the development of sodium-sensitive hypertension in Dahl S rats and the desensitization of arterial baroreflex control of RSNA and heart rate (HR) were prevented by concomitant central administration of antibody Fab fragments.
Four-week-old male inbred Dahl S rats were supplied by Harlan Sprague Dawley, Inc (Indianapolis, Ind) from June to November 1992. 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) and were housed in an animal room (temperature, 24°C; 12-hour light/dark cycle) with free access to tap water. At 5 weeks of age, rats were anesthetized with sodium pentobarbital (65 mg/kg IP), and a 23-gauge, stainless steel, right-angled cannula was implanted into the left brain lateral ventricle (0.5 mm posterior and 1.4 mm lateral to bregma and 3.3 mm deep from dura) and fixed to the skull. The cannula was connected to an osmotic minipump (model 2002, Alza Corp) with PE-50/60 tubing. The pumps were filled with a solution of either antibody Fab fragments (Digibind, Burroughs Wellcome Inc) or γ-globulins (Sigma Chemical Co). Both were infused at 200 μg/12 μL per day for 14 days. In one group of rats on high sodium, the pump filled with the same amount of Fab fragments was connected to a permanent intravenous catheter that was inserted into the right jugular vein with rats under pentobarbital anesthesia. All pumps were implanted subcutaneously at the back of the rats. Original diets were resumed after surgery. Penicillin G (30 000 IU, Derapen, Ayerst Laboratories) was injected intramuscularly after surgery.
At 7 weeks of age, the rats were anesthetized with halothane, and catheters were placed in the right femoral artery and jugular vein. After intravenous injection of methohexital sodium (30 mg/kg Brevital supplemented with 10 mg/kg as needed; Eli Lilly Canada Inc), a pair of platinum electrodes (A-M Systems, Inc) was placed through a flank incision around the left renal nerve and secured with silicone rubber (SilGel 604, Wacker).2 The electrodes and catheters were tunneled subcutaneously to the back of the neck.
Four hours after recovery from anesthesia, each rat was placed in a plastic cage without any restriction of movement. The intra-arterial catheter was connected to a transducer, and BP and HR were recorded through a polygraph (model 7E, Grass Instrument Co) and Grass 7P44 tachograph. An infusion pump (model 355, Sage Instruments) was used for drug administration through the intravenous catheter. The electrodes were linked to a Grass P511 band-pass amplifier, and RSNA was counted through a nerve traffic analyzer (model 706C, University of Iowa Department of Bioengineering). The method of counting suprathreshold electrical activity was used to quantify the nerve activity (spikes per second).8 The output of counts was displayed on the polygraph as a frequency-time histogram together with mean arterial pressure (MAP) and HR. RSNA, MAP, and HR were also digitized by a microcomputer. At the end of an experiment, the rat was killed, and 20 minutes later the background noise of the RSNA recording was measured by directly recording the activity. The actual RSNA was determined by subtracting noise from the total activity.
After a 30-minute stabilization, basal MAP, HR, and RSNA were recorded. Phenylephrine (5 to 50 μg/kg per minute) in 5% dextrose was infused at progressively increasing doses to induce a ramp increase of MAP by 50 mm Hg over 2 to 3 minutes. Twenty minutes after BP, RSNA, and HR had returned to basal levels, nitroprusside (5 to 100 μg/kg per minute) in 5% dextrose was infused to induce a ramp decrease of MAP by 50 mm Hg over 2 to 3 minutes. The infusion speed for both was less than 0.08 mL/min. MAP, HR, and RSNA were recorded continuously throughout the experiment.
The resting RSNA (spikes per second) of each rat was considered as 100%, and responses of RSNA to changes in MAP were expressed as the percentage of resting value in all experiments. RSNA and HR responses were plotted against MAP changes evoked by phenylephrine and nitroprusside infusion. The changes in RSNA or HR in response to changes in MAP were analyzed as a logistic model9 using the logistic equation ΔRSNA=P1+P2/[1+eP3(ΔMAP−P4)], 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, the ΔMAP at half the ΔRSNA range. The average gain (G) or slope of the curve between the two inflection points is given by G=−P2×P3/4.56, and the upper plateau=P1+P2, which represents the maximal increase in RSNA. The curve of best fit was obtained with the Sigmoid program provided by the Baker Medical Research Institute, Victoria, Australia. In addition, changes in RSNA or HR in response to decreases or increases in MAP elicited by intravenous nitroprusside or phenylephrine were analyzed separately. The gains (slopes) of responses were obtained by analyzing the linear portion of the curves by linear regression. For all comparisons, the data were assessed using ANOVA followed by Duncan’s multiple range test. The level of statistical significance was set at 5%.
Table 1⇓ shows resting BP, HR, and body weight gain. MAP was significantly increased by high sodium in rats treated with γ-globulins compared with rats on regular sodium. In contrast, MAP was not increased by high sodium in rats treated with ICV Fab fragments compared with those on regular sodium treated with ICV Fab fragments or γ-globulins. MAP in rats on high sodium treated with intravenous Fab fragments was significantly higher than in those on high sodium with ICV Fab fragments but significantly lower than in those on high sodium treated with γ-globulins. There were no significant differences in resting HR among all rat groups. Body weight gain was slightly less in all rats on high sodium versus the regular sodium controls.
Baroreflex Control of RSNA
An increase or decrease in BP caused inhibitory and excitatory responses of RSNA (Fig 1⇓). RSNA responses in rats on regular sodium treated with Fab fragments and γ-globulins were similar in terms of sensitivity (average gain) and maximal responses (Table 2⇓). In rats treated with γ-globulins, maximal RSNA responses were similar during high versus regular sodium, but the sensitivity was significantly attenuated during high versus regular sodium (Table 2⇓). In contrast, compared with rats on regular sodium and ICV Fab fragments, the sensitivity of the RSNA response in rats on high sodium with ICV Fab fragments was not attenuated but slightly augmented (P=.07, Table 2⇓). The maximal responses were similar in rats on high sodium versus regular sodium treated with Fab fragments.
In rats on high sodium treated with intravenous Fab fragments, the sensitivity of RSNA responses was significantly higher than in rats on high sodium treated with γ-globulins and was similar to that in rats on regular sodium (Table 2⇑).
When changes in RSNA in response to increases and decreases in MAP were analyzed separately by means of linear regression, patterns of changes in baroreflex sensitivity were observed that were similar to those seen by analyzing the data with the logistic equation (Table 3⇓). However, in contrast to the statistical result obtained using logistic analysis, in rats on high sodium with ICV Fab fragments the sensitivity of baroreflex control of RSNA assessed by either increasing or decreasing MAP was significantly augmented compared with rats on regular sodium and ICV Fab fragments (Tables 2⇑ and 3⇓).
Baroreflex Control of HR
Similar to the responses of RSNA, changes in MAP elicited tachycardia and bradycardia. The gain of changes in HR was again significantly attenuated in γ-globulin–treated rats on high versus regular sodium. In rats on high sodium, the gain of HR responses was significantly higher in rats treated with Fab fragments versus γ-globulins and similar to the gain in rats on regular sodium treated with ICV Fab fragments (see Tables 3⇑ and 4⇓ and Fig 2⇓).
In the present and other studies performed in our laboratory during the same time period,7 10 salt-sensitive hypertension was consistently expressed in Dahl S rats. Moreover, other effects such as increased sympathoexcitatory and decreased sympathoinhibitory responses7 as well as increased brain “oubain” content10 were observed in the Dahl S rats on high sodium, indicating that the recently demonstrated genetic contamination in Dahl S rats from Harlan Sprague Dawley11 was not yet a major problem before 1993.
In normotensive rats,6 ICV administration of antibody Fab fragments blocks the sympathoexcitatory and pressor responses to ICV hypertonic saline. In Dahl S rats, high dietary sodium increases brain “oubain” content,10 and ICV Fab fragments prevent and reverse sodium-induced hypertension.7 In SHR, brain “oubain” content is also increased by high sodium intake,12 and ICV Fab fragments prevent the sodium-induced exacerbation of hypertension.13 These observations suggest that increased release of brain “oubain” mediates the sympathoexcitatory and hypertensive effects of high sodium intake in these rats. The major new information provided in the present study is that in Dahl S rats, sodium-induced desensitization of arterial baroreflex function can be prevented by concomitant central administration of antibody Fab fragments.
High sodium induces different changes in arterial and cardiopulmonary baroreflex function in normotensive versus hypertensive rats, ie, it sensitizes baroreflex function in normotensive rats1 2 3 but does not sensitize,1 3 or even desensitize,4 the reflex control in genetically hypertensive rats. Sensitization of arterial baroreflex control of RSNA tends to restrain RSNA and induce natriuresis, which can compensate for the high sodium–induced volume expansion and thus prevent an increase in BP.14 Desensitization or lack of sensitization of arterial baroreflex control of RSNA does not restrain the RSNA in response to volume expansion and thus fails to cause natriuresis and prevent the rise of BP. In support of this concept, high sodium intake induces hypertension in normotensive rats after chronic sinoaortic denervation.1 2 In intact Dahl S rats or SHR on high sodium, the sensitization is absent, and the hypertension is exacerbated but not further exaggerated after chronic sinoaortic denervation.1
Both peripheral and central mechanisms could be involved in the high sodium–induced changes in the arterial baroreflex. Ferrari and Mark3 showed that in anesthetized Dahl R but not in Dahl S rats, high sodium potentiated afferent arterial baroreceptor function, assessed by enhanced afferent aortic depressor nerve activity in response to phenylephrine-elicited increase in BP. Compared with Dahl R rats or Dahl S rats on regular sodium, in anesthetized Dahl S rats on high sodium, changes in both afferent aortic depressor nerve activity and efferent splanchnic sympathetic nerve activity in response to changes in BP were attenuated,4 but decreases in splanchnic4 and lumbar15 sympathetic nerve activities induced by electrical stimulation of the aortic depressor nerve were not altered, suggesting the absence of baroreflex modulation at the central level. However, bradycardia elicited by electrical stimulation of the aortic depressor nerve was attenuated in anesthetized Dahl S rats on high sodium.4 Thus, in addition to peripheral effects causing desensitization, high sodium may also act centrally to desensitize arterial baroreflex function in Dahl S rats.
The actual mechanisms of sodium-induced changes in baroreflex sensitivity are still unclear. Buñag and Miyajima5 showed that long-term ICV hypertonic saline causes impairment of baroreflex function and later hypertension in normotensive rats. Short-term ICV hypertonic saline, ouabain, or brain extracts containing endogenous ouabain induce similar sympathoexcitatory and pressor effects in conscious rats6 ; all of these effects can be blocked by pretreating the rats with ICV antibody Fab fragments. We hypothesized1 that the increase in brain “oubain” induced by a high sodium intake may contribute to the different changes in arterial baroreflex sensitivity in SHR versus WKY. Since the increase in brain “oubain” is more marked in SHR12 and Dahl S rats10 than in their normotensive controls, we proposed that in these substrains, central effects of “oubain” are different and/or more marked and may obscure or overcome the possible sensitization of the arterial baroreflex by peripheral “oubain.”16 17 The present study supports this hypothesis, because ICV Fab fragments concomitantly with high sodium intake prevented desensitization of the arterial baroreflex and even caused sensitization (Table 3⇑) in Dahl S rats on high sodium. These observations indicate that in Dahl S rats on high sodium, centrally administered Fab fragments block brain “oubain” and thereby prevent the desensitization of the baroreflex but may not affect the increase in peripheral “oubain,” which can sensitize the arterial baroreflex. Both changes are in favor of decreasing BP. Compared with the effects of centrally administered Fab fragments, peripherally administered Fab fragments resulted in less-effective prevention of the increase in BP. The intravenous dose used in the present study may affect central “oubain” possibly to a less extent. Alternatively, or in addition, the intravenously administered Fab fragments probably blocked peripheral “oubain,” thereby preventing sensitization of the arterial baroreflex peripherally.
In the present study, reflex control was evaluated in conscious Dahl S rats. Desensitization by high sodium was prevented by ICV Fab fragments, suggesting sodium-induced desensitization of reflex control of RSNA in the central nervous system. In contrast, in anesthetized Dahl S rats on high sodium, no desensitization of baroreflex control of splanchnic or lumbar sympathetic nerve activities was detected at the central level, assessed by electrical stimulation of the aortic depressor nerve.4 15 Several factors may contribute to the discrepancy between the present and previous findings. First, general anesthesia affects arterial baroreflex function18 and may mask central effects of high sodium. Second, the central nervous system controls differently sympathetic outflow to different organs and tissues.8 19 Third, electrical stimulation of the aortic depressor nerve is frequency dependent in salt-sensitive hypertension20 : in SHR, low-frequency stimulation activates only part of the central reflex control, which is salt insensitive, whereas activation of the salt-sensitive part of the baroreflex control requires higher-frequency stimulation. The latter was not evaluated in earlier studies.4 15
In addition to brain “oubain,” other agents such as angiotensin II may act centrally to desensitize the arterial baroreflex.21 In studies in vitro, Fab fragments bind oubain, human “oubain,”22 and rat brain “oubain”10 specifically with high affinity. In vivo,6 they block sympathoexcitatory and pressor responses elicited by centrally administered ouabain or brain “oubain” but not by central angiotensin II or carbachol, supporting the specificity of the Fab fragments. It is therefore unlikely that the blunted baroreflex desensitization in Dahl S rats on high sodium resulted from the blockade of brain angiotensin II by Fab fragments.
In the present study, the possibility cannot be ruled out that prevention of the desensitization of arterial baroreflex control of RSNA and HR in Dahl S rats is the result of prevention of the hypertension rather than blockade of a specific brain “oubain” mechanism. In renal hypertensive rats,23 short-term unclipping reverses hypertension and causes rapid normalization of the desensitized peripheral component of the arterial baroreflex. The authors suggested that this desensitization may be secondary to the changes in resting BP. However, after the increase in BP was prevented by 6-OH-dopamine, the absence of baroreflex sensitization still remained in Dahl S rats on high sodium.3 Moreover, desensitization of the arterial baroreflex in Dahl S rats has been shown even before the high sodium diet is begun.15 24
In conclusion, sodium-induced hypertension in Dahl S rats is associated with a desensitization of arterial baroreflex control of RSNA and HR. Blockade of brain “oubain” with antibody Fab fragments abolishes the desensitization and prevents the development of sodium-sensitive hypertension. These results support the concept that as one of the mechanisms of sodium-sensitive hypertension, a sodium-induced increase in brain “oubain” desensitizes the arterial baroreflex, thereby facilitating the development of hypertension in Dahl S rats.
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.
- Received June 23, 1994.
- Revision received August 16, 1994.
- Accepted November 7, 1994.
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