(Hypertension. 1999;34:729-732.)
© 1999 American Heart Association, Inc.
Scientific Contributions |
L-Arginine Restores the Effect of Ouabain on Baroreceptor Activity and Prevents Hypertension
From the Physiological Sciences Graduate Program, Biomedical Center, Federal University of Espirito Santo, Vitoria, ES, Brazil
Correspondence to Elisardo C. Vasquez, PhD, Physiological Sciences Graduate Program, Biomedical Center, UFES, Av. Marechal Campos 1468, 29040–090 Vitoria, ES, Brazil. E-mail evasquez{at}zaz.com.br
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Abstract |
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Abstract—In spontaneously hypertensive rats, ouabain exerts an excitatory effect on baroreceptor nerve activity (BNA). The aim of this study was to determine the effects of ouabain on BNA in other experimental models of hypertension and its interaction with nitric oxide. Rats were made hypertensive using the procedures for N
-nitro-L-arginine methyl
ester (L-NAME), deoxycorticosterone acetate (DOCA) salt, and 2-kidney,
1 clip (2K1C) hypertension models. In these groups, systolic
arterial pressure was 195±7, 149±6, and 148±4
mm Hg, respectively, compared with 110±4 mm Hg in normotensive
rats. Acute ouabain administration had an excitatory effect on BNA in
normotensive rats (37±4%), an inhibitory effect in L-NAME
hypertensive rats (-60±7%), and no effect in DOCA-salt and 2K1C
hypertensive rats. The effects of ouabain were not related to
arterial pressure levels, and no excitatory effect on BNA
was observed in prehypertensive DOCA-salt rats. Long-term
administration of L-arginine (3 g ·
kg-1 · day-1) prevented DOCA-salt
(121±8 mm Hg) and 2K1C (104±4 mm Hg) hypertension,
markedly attenuated L-NAME (130±9 mm Hg) hypertension, and
restored the excitatory effect of ouabain on BNA in these groups to
levels similar to the normotensive rats and their respective control
groups. We conclude that ouabain has a diverse effect on BNA in
experimental models of hypertension, and it can be normalized by
L-arginine. The data also indicate that nitric oxide may
play a pivotal role in mediating the excitatory effect of ouabain on
BNA, and we speculate that a therapeutic combination of ouabain and
L-arginine may be beneficial in secondary
hypertension.
Key Words: arginine • ouabain • L-NAME • baroreceptors • hypertension
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Introduction |
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The baroreflex acts to impede short-term fluctuations in arterial pressure (AP),1 but it is impaired in pathological states such as long-term hypertension.2 Although mechanical deformation of the nerve endings is considered the primary mechanism of baroreceptor activation, baroreceptor sensitivity is modulated by endothelial factors, such as nitric oxide (NO), which is known to inhibit baroreceptor nerve activity (BNA).3 However, ionic mechanisms, including the inhibition of Na+-K+-ATPase pump activity, increase BNA.4 5 Ouabain, which is known as an inhibitor of Na+-K+-ATPase pump activity, reportedly has an excitatory effect on BNA.6 7
Recently, we reported that the excitatory effect of ouabain on BNA is
increased in spontaneously hypertensive rats.7 The purpose
of the present study was to determine whether ouabain has an
excitatory effect on BNA in deoxycorticosterone acetate (DOCA) salt and
2-kidney, 1 clip (2K1C) hypertensive rats. Because we did not observe
such an effect in these models and because the high plasma volume could
elicit greater vascular shear stress, which is a stimulus for
NO,8 we tried to restore the excitatory effect of ouabain
on BNA in these models of hypertension by treating the animals with
L-arginine long term. Additionally, we evaluated the
effects of ouabain on BNA in
N-nitro-L-arginine
methyl ester (L-NAME) hypertensive rats.
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Methods |
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Animal
The experimental protocols were performed in male Wistar rats. All experiments were conducted in compliance with the guide for biomedical research, as stated by the Brazilian Societies of Experimental Biology, and with the guiding principles of other physiological societies for research involving animals.
DOCA-Salt Hypertension
As previously described,9 45-day-old rats were
anesthetized with urethane (1.2 mg/kg IP) and
uninephrectomized; 4 days later, they were treated with either DOCA
(Sigma; 8 mg/kg SC) or vehicle (soybean oil, 0.25 mL/rat SC; n=8). DOCA
was administered twice a week for 20 days (prehypertensive group; n=8)
or 40 days (hypertensive group; n=8). DOCA-treated rats were allowed
free access to water containing 1% NaCl and 0.03% KCl. Control
animals were treated with DOCA and allowed free access to water without
the electrolyte mixture (DOCA-water; n=8).
L-NAME–Induced Hypertension
This model of hypertension was induced as previously
described.10 11 Briefly, rats (250 to 300 g; n=8)
were housed in individual cages and treated with L-NAME (Sigma)
dissolved in water (0.5 mg/mL; 11±2 mg/day) for 2 days. Each age- and
body weight–matched control rat (n=8) was given water only (volume
equal to that consumed by the respective hypertensive paired rat).
Renovascular Hypertension
2K1C renovascular hypertension was obtained by applying a silver
clip with an internal diameter of 0.2 mm to the left renal artery
in 45-day-old rats, under ether anesthesia, as previously
described.2 Control sham-operated rats were subjected to
isolation, but not constriction, of the renal artery. Both 2K1C (n=8)
and control (n=8) animals were studied 30 days later.
L-Arginine Treatment
Age-matched animals (n=8 per group) subjected to DOCA-salt (20
and 40 days), 2K1C, and L-NAME hypertension were
simultaneously treated with the NO precursor
L-arginine (Sigma) dissolved in water (3 g ·
kg-1 · day-1). The
respective control groups (n=8 per group) were given water only.
Surgical Procedures for Baroreceptor Nerve Recording
After a ventral midline neck incision, the paratracheal muscles
were retracted, and a tracheotomy was performed to permit the animals
to breathe freely. The recurrent laryngeal nerve identified near the
trachea was chosen because it displays a markedly afferent
BNA.6 12 After the confirmation of whole-nerve activity,
the ipsilateral vagus nerve was sectioned proximally to eliminate any
respiratory-related activity on the recurrent laryngeal nerve.
Pulsatile AP was recorded through a polyethylene cannula inserted
into the femoral artery and connected to a pressure transducer. The
femoral vein was cannulated for drug injections. The recurrent
laryngeal nerve was isolated and placed on a silver bipolar electrode.
Extracellular action potentials were recorded with an AC amplifier
(NL 104, NeuroLog, Digitimer). The amplified signals were filtered,
connected to an audio amplifier, displayed on an oscilloscope
(Tektronix), and stored for subsequent analysis (Biopac Inc).
The data were processed with a spike trigger and a ratemeter, and they
were displayed on polygraph (Gould). All data were digitized and stored
for further analysis.
Experimental Protocol
After the preparation was stabilized for 30 minutes, BNA and AP
were recorded under basal conditions for 15 minutes. Next, the
functional integrity of the baroreceptor nerve afferent was tested by
recording the changes in BNA during increases and decreases of
AP induced by phenylephrine (1 µg/kg IV) and sodium
nitroprusside (0.1 µg/kg IV), respectively. After recovering the
basal values of BNA and AP, ouabain (30 µg IV) was injected, and the
changes in systolic AP and BNA were quantified 30 minutes
later.
Statistical Analysis
Systolic AP and BNA baseline values after ouabain
administration were compared among groups by use of a 2-factor ANOVA
followed by a Tukey's post-hoc test. Differences were considered
significant at P<0.05.
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Results |
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Effects of Long-Term Administration of L-Arginine on AP
Table 1 shows the baseline values of systolic and diastolic AP in hypertensive rats and their respective controls. Administration of L-NAME for 2 days caused the highest levels of both systolic and diastolic AP compared with other models of hypertension. Moderate hypertension was observed in rats treated with DOCA-salt for 40 days, but not in those treated for 20 days only, when compared with DOCA-water control rats. The 2K1C group showed moderate hypertension when compared with the control group. L-arginine treatment did not have any effect on AP in control rats or in rats treated with DOCA-salt for 20 days (prehypertensive). However, L-arginine treatment for 40 days prevented hypertension in 2K1C and in DOCA-salt 40-day rats. Rats treated with L-NAME plus L-arginine for 2 days showed marked attenuation (P<0.01) in the development of hypertension.
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Effects of Ouabain on AP and BNA
Ouabain did not change the baseline values of systolic or
diastolic AP in normotensive, hypertensive (DOCA-salt for
40 days, 2K1C, and L-NAME groups), or L-arginine-treated
hypertensive rats (Table 1). As illustrated in Figure 1 and summarized in Figure 2, after ouabain injection, a similar
increase in BNA occurred in both normotensive (37±4%) and control
groups: DOCA-water for 20 days (38±10%), DOCA-water for 40 days
(39±10%), sham-operated 2K1C (38±5%), and L-NAME vehicle-treated
(36±4%). No excitatory effect on BNA was observed with the short-term
administration of ouabain in 2K1C (2±3%) or DOCA-salt for 40 days
(2±10%) hypertensive rats or DOCA-salt for 20 days (-5±9%)
normotensive rats (Figure 2B). In contrast to normotensive rats
(37±4%) and the hypertension models, ouabain had an
inhibitory effect on BNA in L-NAME rats (-60±7%). As
summarized in Figure 2B, long-term treatment of hypertensive
rats with L-arginine prevented ouabain's
inhibitory effect on BNA in DOCA-salt for 40 days
(48±7%), 2K1C (42±5%), and L-NAME (15±8%) hypertensive animals
and in DOCA-salt for 20 days prehypertensive animals (37±7%). The
excitatory effect of ouabain on BNA in these groups treated with
L-arginine was similar to that observed in control groups
(Figure 2).
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Discussion |
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The present study had 3 major findings: (1) ouabain inhibits BNA in L-NAME hypertensive rats but has no effect in DOCA-salt and 2K1C hypertensive rats; (2) prehypertensive DOCA-salt rats do not show the ouabain-induced excitatory effect on BNA, suggesting that such an effect is not related to AP levels; and (3) long-term administration of L-arginine prevents hypertension and restores the excitatory effect of ouabain on BNA.
In contrast to our previous observations in spontaneously hypertensive rats,7 in the present study, we observed that ouabain injection did not increase BNA in DOCA-salt and 2K1C hypertensive rats. A reasonable explanation for the enhanced response of spontaneously hypertensive rats to ouabain could be an active mechanism of production of ouabain-like substances in this strain.13 However, high levels of ouabain have also been observed in DOCA-salt hypertensive rats,14 and we did not observe an excitatory effect of ouabain on BNA in this model at either the prehypertensive (20 days) or hypertensive (40 days) stage. This could not be attributed to DOCA alone because ouabain, when injected into DOCA-water rats, increased BNA to the same levels observed in normotensive rats. Interestingly, the excitatory effect of ouabain on BNA in the control groups and the lack of effect in 2K1C and DOCA-salt groups was pressure-independent; no changes in resting AP were observed in those groups.
Hypertension in long-term 2K1C rats has been attributed to a combination of elevated angiotensin II and high plasma volume,15 and the latter factor is a determinant in the DOCA-salt model as well.14 Thus, one could hypothesize that in these models of hypertension, greater vascular shear stress, which is a stimulus for endothelium-derived factors,8 increased the synthesis of NO, which is known to suppress BNA.3 In addition, the increased levels of NO in 2K1C may counterbalance the vasoconstrictor effect of angiotensin II.16 We did not test this hypothesis because the blockade of NO synthesis with L-NAME actually decreased BNA when the effects of ouabain were evaluated. On the other hand, L-arginine long-term treatment restored BNA in 2K1C and DOCA-salt groups to the same levels as normotensive rats, but it did not amplify the excitatory effect of ouabain on BNA in normotensive rats. This finding indicates that augmentation of NO synthesis could be necessary for an effective inhibitory action of ouabain on the Na+-K+-ATPase pump in baroreceptor endings and, thus, the BNA of DOCA-salt and 2K1C rats would increase. If the production of NO is impaired in arterial hypertension, as has been reported,17 it would explain the lack of effect of ouabain in these models of hypertension.
We speculate that the lack of effect of ouabain on BNA in 2K1C and DOCA-salt rats could be due to inadequate amounts of NO. Interestingly, ouabain caused an inhibitory effect on BNA in L-NAME-treated rats, suggesting that NO is essential for the inhibitory action of ouabain in the Na+-K+ pump and/or that ouabain has a secondary ionic action on baroreceptors. The reversal effect of ouabain on BNA in L-NAME rats treated with L-arginine, although the AP was not completely normalized, is another point in favor of the hypothesis that ouabain is partially dependent on NO for its inhibitory action on the Na+-K+-ATPase pump, thus acting as an excitatory agent of baroreceptor endings.
Although additional experiments are necessary to explain the mechanism of interaction of NO with ouabain, we speculate that ouabain, in addition to its direct cardiotonic effect, reduces efferent sympathetic activity through the activation of BNA.
Received May 8, 1999; first decision June 15, 1999; accepted June 20, 1999.
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References |
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TopAbstractIntroductionMethodsResultsDiscussionReferences |
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- Vasquez EC, Meyrelles SS, Mauad H, Cabral AM. Neural reflex regulation of arterial pressure in pathophysiological conditions: interplay among the baroreflex, the cardiopulmonary reflexes and the chemoreflex. Braz J Med Biol Res. 1997;30:521–532.[Medline] [Order article via Infotrieve]
- Moysés MR, Cabral AM, Vasquez EC. Time course changes in cardiac baroreflex in conscious renovascular hypertensive rats. Hypertension. 1994;23:I87–I92.
-
Matsuda T, Bates JN, Lewis SJ, Abbout FM, Chapleau MW.
Modulation of baroreceptor activity by nitric oxide and
S-nitrosocysteine. Circ Res. 1995;76:426–433.
[Abstract/Free Full Text] -
Chapleau MW, Hajduczok G, Abboud F. Paracrine
modulation of baroreceptor activity by vascular
endothelium. News Physiol Sci. 1991;6:210–214.
[Abstract/Free Full Text] - Chapleau MW, Abboud F. Modulation of baroreceptor activity by ionic and paracrine mechanisms: an overview. Braz J Med Biol Res. 1994;27:1001–1015.[Medline] [Order article via Infotrieve]
-
Chapleau MW, Lu J, Hajduczok G, Abbout FM. Mechanism
of baroreceptor adaptation in dogs: attenuation of adaptation by the
K+ channel blocker
4-aminopyridine. J Physiol. 1993;462:291–306.
[Abstract/Free Full Text] - Abreu GR, Cabral AM, Futuro Neto HA, Vasquez EC. Ouabain produces diverse excitatory effects on afferent baroreceptor nerve activity in SHR and WKY animals. Clin Exp Hypertens. 1998;20:85–94.
-
Vanhoutte PM. Endothelium and control
of vascular function. Hypertension. 1989;13:658–667.
[Abstract/Free Full Text] - Bissoli NS, Vasquez EC, Cabral AM. Chlorthalidone-modulated vascular responses to vasodilator stimuli in DOCA-salt hypertensive rats. Pharmacol Res. 1996;33:47–54.[Medline] [Order article via Infotrieve]
- Cunha RS, Cabral AM, Vasquez EC. Evidence that the autonomic nervous system plays a major role in the L-NAME-induced hypertension in conscious rats. Am J Hypertens. 1993;6:806–809.[Medline] [Order article via Infotrieve]
- Araujo MTM, Barker LA, Cabral AM, Vasquez EC. Inhibition of nitric oxide synthase causes profound enhancement of the Bezold-Jarisch reflex. Am J Hypertens. 1998;11:66–72.[Medline] [Order article via Infotrieve]
- Krieger EM, Vasquez EC, Trindade AS Jr. Heart rate changes in neurogenic hypertension. In: Szentiványi M, Juhász-Nagy A, eds. Factors Influencing Adrenergic Mechanisms in the Heart: Satellite Symposium of the 28th International Congress of Physiological Sciences. Vol 27. New York: Pergamon Press; 1981:11–19.
-
Doris PA. Ouabain in plasma from spontaneously
hypertensive rats. Am J Physiol. 1994;266:H360–H364.
[Abstract/Free Full Text] - Semplicini A, Serena L, Valle R, Ceolotto G, Felice M, Fontebasso A, Perssina AC. Ouabain inhibiting activity of aldosterone antagonists. Steroids. 1995;60:110–113.[Medline] [Order article via Infotrieve]
- Carretero OA, Gulati OP. Effects of angiotensin antagonist in rats with acute, subacute and chronic two-kidney renal hypertension. J Lab Clin Med. 1978;91:264–271.[Medline] [Order article via Infotrieve]
-
Sigmon DH, Beierwaltes WH. Influence of nitric oxide in
the chronic phase of two-kidney, one clip renovascular hypertension.
Hypertension. 1998;31:649–656.
[Abstract/Free Full Text] - Higashi Y, Oshima T, Ono N, Higara H, Yoshimura M, Watanabe M, Matsura H, Kambe M, Kajiyama G. Intravenous administration of L-arginine inhibits angiotensin-converting enzyme in humans. J Clin Endocrinol Metabol. 1995;80:2198–2202.[Abstract]
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