INTRODUCTION

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HIGH DIETARY SODIUM CHLORIDE CAN HAVE an important influence on arterial pressure (10, 16, 45). The NaCl-dependent pressor response is thought to be due, at least in part, to a compromised ability of the kidneys to excrete sodium (45) and may be mediated by genetic and/or environmental factors (14, 18).

Endogenous digitalis-like sodium pump ligands (SPL) are thought to link sodium/fluid retention to regulation of blood pressure (10, 17, 16, 19, 20). It has been hypothesized that the primary role of endogenous SPL is to promote natriuresis via inhibition of sodium reabsorption in the renal proximal tubules (6). However, in addition to this effect, increased plasma concentrations of SPL can contribute to vasoconstriction via inhibition of the Na-K pump coupled with activation of Na⁺/Ca²⁺ exchange in vascular smooth muscle (6, 20). In support of this view, plasma levels of SPL correlate with the level of blood pressure in both human (20) and rat (44) hypertension.

Several subtypes of endogenous SPL have been detected in mammals; among them a cardenolide, an ouabainlike compound (OLC) (30, 42), and bufadienolides (2, 28, 40), including the marinobufagenin (MBG)-like factor (2, 3). MBG exhibits vasoconstrictor activity in vitro (4) and is responsive to plasma volume expansion in dogs and rats (3, 11). Increases in the plasma concentration of MBG accompany the blood pressure elevations during voluntary hypoventilation by normotensive humans (2), behaviorally induced pressor responses in micropigs (13), adrenocorticotropin-induced hypertension in rats (11), and in patients with preeclampsia (29). In acute plasma volume-expanded rats, MBG increases in plasma, whereas OLC increases in the pituitary (11). In several studies, brain OLC was responsive to various stressor stimuli (15, 23, 24).

Both high salt intake and psychosocial stress are thought to mediate the elevations in blood pressure (14, 18, 22). Stress has been shown to potentiate salt sensitivity of blood pressure, and high NaCl intake enhances the elevation of arterial pressure in response to stress. Thus a combination of behavioral conditioning and high salt intake produces sustained pressor responses in dogs, whereas neither behavioral conditioning nor high salt intake per se increased blood pressure (1). Subsequently, psychosocial stress was found to facilitate the development of salt-induced hypertension in baboons (41) and in humans (9).

Thus the evidence indicates that either high dietary NaCl or behavioral stress alone can stimulate the production of SPL and contribute to hypertension. The purpose of our study was to investigate the links between SPL and blood pressure in normotensive rats exposed to high NaCl intake and behavioral stress alone and in combination.

	METHODS

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General. The protocol of the study has been approved by the Institutional Animal Care and Use Committee of the Gerontology Research Center, National Institute on Aging. Thirty-two male, 6-mo-old Fisher 344 × Norwegian brown (F344 × NB) rats (408 ± 10 g) were obtained from Harlan Sprague Dawley (Indianapolis, IN). The experiments were begun after an adaptation period of 7 days. All rats were kept at 26°C with a 12:12-h light-dark cycle on a normal (0.5%)- or high (4%)-NaCl diet (both supplied by ICN Biochemicals, Cleveland, OH) and tap water ad libitum. For the subsequent 35-day experimental period, body weights (BW), systolic blood pressure (SBP), water consumption, and urine output were measured weekly. SBP was recorded in conscious animals by tail-cuff plethysmography (IITC model 31 IITC Life Science). Twenty-four-hour urine samples were filtered through Millipore filters (0.45 µm, Millipore Products Division, Bedford, MA) and kept frozen (80°C) for measurement of Na⁺, K⁺, OLC, and MBG. The animals were divided into four experimental groups (n = 8 each):

Control group. Rats were kept in groups of four in regular cages on a normal NaCl diet (0.5%) and were individually placed in metabolic cages on days 0, 1, 5, 15, 25, and 35 of the experiment.

NaCl-loaded rats (Salt group). Rats were kept in groups of four in regular cages on a high-NaCl diet (4%) and were individually placed in metabolic cages as above.

Socially isolated rats (Iso group). For 35 consecutive days, rats were isolated individually in standard 23-cm diameter and 13-cm-high circular metabolic cages. Each cage was isolated from others by sound- and viewproof plastic. Rats were further isolated from the equipment and experimenters in larger chambers (100 × 150 × 230 cm). The temperature was maintained at 26°C, the light-dark cycle was 12:12 h. Water was available ad libitum, and the animals received a normal (0.5% NaCl) diet.

Combination of isolation and NaCl loading (Iso+Salt group). Rats were isolated as in the Iso group but received a 4% NaCl diet.

Tissue weights. At 35 days, rats from all groups were anesthetized by intraperitoneal injection of 60 mg/kg pentobarbital sodium and killed by exsanguination from the abdominal aorta. Plasma samples were frozen for measurement of MBG and OLC. Kidneys, heart, and thoracic aorta were removed immediately, blotted, and weighed. Wet organ weights were expressed in grams per 100 g of body weight.

Ouabain- and MBG-like immunoreactivity. Plasma and urinary samples (0.5 ml) were applied to Sep-Pak C₁₈ cartridges that had been activated with acetonitrile. After washing with 10 ml distilled water, OLC and MBG were eluted with acetonitrile (7.5 ml 20% acetonitrile followed by 7.5 ml of 80% acetonitrile). The eluate was evaporated and reconstituted in the initial volume with assay buffer. The MBG immunoassay was performed as recently described (3). The assay is based on competition between immobilized antigen (MBG-glycoside-RNAase) and SPL within the sample for a limited amount of binding sites on polyclonal rabbit MBG (raised against MBG-glycoside-BSA) antibody (1:6,000). Secondary (goat anti-rabbit) antibody (1:2,000; Sigma Chemicals, St. Louis, MO) was labeled with europium (Wallac-Oy labeling kit, Turku, Finland). The cross-reactivity of the MBG antibody was (in %): 100 MBG, 0.1 ouabain, 1.0 digoxin, 3.0 digitoxin, 1.0 bufalin, 1.0 cinobufagin, <0.1 prednisone, <0.1 spironolactone, <1.0 proscillaridin, <0.1 progesterone, and <5 mixture of bufadienolides from Bufo marinus venom except MBG. The OLC assay was based on a similar principle utilizing an ouabain-ovalbumin conjugate and rabbit ouabain antibody (1:100,000; Chemicon International, Temecula, CA). The cross-reactivity of the ouabain antibody is (in %): 100 ouabain, 7.4 digitoxin, <0.01 progesterone, <0.01 5-beta cholanic acid, prednisone, and canrenoic acid, 0.2 proscillaridin, 0.26 MBG-free mixture of bufadienolides from Bufo marinus toad venom, 0.03 bufalin, 0.09 aldosterone, and 0.5 MBG.

Urinary electrolytes. Urinary Na⁺ and K⁺ concentrations were measured using the ion-electrode technique (Beckman Instruments, Synchron, EL/ISE), and total electrolyte excretion was expressed as millimoles per 24 h.

Statistics. Results are expressed as means ± SE and analyzed statistically using one-way ANOVA followed by Bonferroni test (intragroup comparisons of repeated measurements), repeated-measures ANOVA followed by Dunnett's test (intergroup comparisons of repeated measurement), and by one-way ANOVA followed by Bonferroni test (intergroup comparisons of nonrepeated measurements; GraphStat Prism, GraphStat, San Diego, CA). Pearson correlation coefficients were determined to assess univariate relationships between selected variables.

Miscellaneous. Chemicals were obtained from Sigma Chemicals.

	RESULTS

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No change in SBP, urinary SPL excretion, or other measured parameters occurred in the control group over the 35 experimental days.

In the Salt group (4% NaCl diet), SBP did not change over 35 days (Fig. 1A). No change in OLC excretion (Fig. 1B) was observed in this group. However, 24-h MBG excretion (Fig. 1C) exhibited gradual increases to levels twice those of baseline (26.0 ± 2.5 pmol/24 h on day 35 vs. 14.6 ± 1.0 pmol/24 h in baseline, P < 0.01). Gradual increases in water intake, urine volume, and urinary excretion of Na and K were also observed in rats in this group (Table 1).

View larger version (23K): [in this window] [in a new window]   Fig. 1.   Changes in systolic blood pressure (SBP; A), 24-h renal excretion of ouabainlike compound (OLC; B), and marinobufagenin (MBG; C) in control, NaCl diet (Salt), isolated (Iso), and Iso+Salt groups. Arrows indicate the beginning of isolation and high-NaCl diet. One-way ANOVA: SBP, control group F = 0.22, P > 0.8; Iso F = 0.11, P > 0.8; Salt F = 0.34, P > 0.8; Iso+Salt F = 5.73, P < 0.001. MBG, control group F = 0.21, P > 0.8; Iso F = 2.18, P = 0.08; Salt F = 5.69, P = 0.0006; Iso+Salt F = 42.7, P < 0.0001. OLC, control group F = 0.53, P = 0.75; Iso F = 15.5, P < 0.0001; Salt F = 2.43, P = 0.05; Iso+Salt F = 3.04, P = 0.019. *P < 0.05, **P < 0.01, vs. baseline, Bonferroni test. Repeated-measures ANOVA followed by Dunnett's test: SBP, Iso+Salt vs. control, P < 0.05; MBG, Salt vs. control, P < 0.05; Iso + Salt vs. control, P < 0.01; OLC, Iso vs. control, P < 0.01; Iso+Salt vs. control, P < 0.05.

The Iso group did not exhibit a change in SBP (Fig. 1A) or in MBG excretion (Fig. 1C). However, urinary OLC excretion showed a threefold peak increase at day 1 (32.0 ± 2.0 vs. 10.0 ± 1.5 pmol/24 h in baseline), then decreased to a level twice that of baseline, and remained at that level for the duration of study (Fig. 1B). Increased diuresis, urinary excretion of Na⁺ and K⁺, and increased water intake also occurred in Iso (Table 1).

The Iso+Salt group exhibited a 9-mmHg increase in SBP compared with baseline values (Fig. 1A). MBG excretion (Fig. 1C) increased by day 5, doubled by day 25, and attained a fourfold increase above baseline at day 35. OLC excretion peaked at day 1 (25.0 ± 2.5 vs. 10.0 ± 2.0 pmol/24 h in baseline), then decreased (17.2 ± 2.7 pmol/24 h, day 5), and subsequently exhibited a secondary sustained increase (Fig. 1B). The patterns of urine excretion and urinary excretion of Na⁺ and K⁺ in the Iso+Salt group were similar to those of the Salt group (Table 1).

As presented in Fig. 2, differences in plasma levels of OLC and MBG at day 35 of the experiment in four experimental groups reflected those observed for renal excretion of both SPL. Compared with control group, plasma MBG concentrations were increased in Salt and Iso+Salt groups, whereas OLC levels were elevated in Iso and Iso+Salt groups.

View larger version (37K): [in this window] [in a new window]   Fig. 2.   Plasma levels of OLC (A) and MBG (B) at day 35 of experiment in rats from control, Iso, Salt, and Iso+Salt groups. Means ± SE. One-way ANOVA followed by Bonferroni test, *P < 0.05, **P < 0.01 vs. control group.

As illustrated in Table 2, heart and kidney absolute and relative (organ wt-to-body wt ratio) weights were increased in both Salt and Iso+Salt groups. Relative aortic weights were increased in Iso and Iso+Salt groups. Over the study period, increases in body weight in control, Salt, and Iso groups were equivalent, but the increase in body weight in the Iso+Salt group was less than that in the three other groups.

Univariate correlations between changes in the excretion of MBG (day 35 vs. day 0 of the experiment) and OLC (day 1 vs. day 0 of the experiment and day 35 vs. day 0 of the experiment) and other parameters in each rat of all four experimental groups combined are presented in Table 3. Plasma levels and changes in the excretion of OLC and MBG were not correlated with the magnitude of changes in SBP. Plasma levels of MBG and OLC positively correlated with renal excretion of these SPL. The relative weights of hearts and kidneys correlated positively with the changes in excretion of MBG, 24-h MBG excretion at day 35, plasma MBG, and changes in sodium excretion but not with the changes in OLC. Aortic weights correlated positively with the changes in urinary excretion of OLC (day 1 vs. day 0, but not day 35 vs. day 0), plasma OLC, 24-h OLC excretion at day 35, and with the changes in MBG and potassium excretion. Neither heart nor kidney weight correlated with the change in SBP. The change in sodium excretion correlated with change in MBG, plasma MBG, but not of OLC excretion. The change in potassium excretion correlated with change in OLC excretion. Neither heart weight nor kidney weight correlated with aortic weight.

	DISCUSSION

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The major finding of the present experiment is that the combination of isolation stress and high NaCl intake in normotensive rats increased renal excretion and plasma levels of two endogenous SPL, OLC, and MBG and caused a modest elevation of SBP. Although high NaCl intake and social isolation per se did not increase SBP, NaCl stimulated MBG, and isolation stress stimulated OLC.

Our results indicate that rats from Iso and Iso+Salt groups exhibited comparable OLC increases, whereas those from Salt and Iso+Salt groups increased MBG plasma levels and renal excretion. Thus it appears that in F344 × NB rats, MBG is more responsive to a high NaCl intake, whereas OLC is more responsive to the isolation stress. Although in some previous reports OLC was shown to be responsive to high NaCl intake in normotensive rats (7, 49), the results of other studies indicate that OLC is not stimulated by high NaCl or that it is responsive to high NaCl intake in animals that exhibit the salt-sensitive blood pressure increases (5, 46, 48). Thus Yamada and co-workers (48), using a highly specific ouabain antibody, demonstrated that although Wistar rats did not exhibit increases in plasma OLC levels during high NaCl intake, such increases did occur in NaCl-loaded Wistar rats after renal mass reduction. Furthermore, Ludens and co-workers (31) demonstrated that increases in SPL in saline-loaded dogs are due to a factor that differs from OLC. Subsequently, saline volume expansion of dogs and Wistar rats was found to be associated with increases in plasma levels of MBG rather than OLC (3, 12).

In previous studies, brain OLC was shown to participate in cardiovascular responses to air stress (24) and swimming stress (15) in normotensive rats and to contribute to the onset of NaCl-induced hypertension in Dahl salt-sensitive rats via centrally mediated sympathoexcitation (23). Furthermore, central administration of ouabain to rats induces persistent hyperactivity (38). That the sympathetic nervous system plays an important role in cardiovascular responses to stress is widely accepted (14). Brain SPL were demonstrated to enhance catecholamine release (37). Conversely, brain catecholamines can modulate levels of brain digitalis-like material, and in rats, central sympathectomy is associated with decreases in hypothalamic and plasma levels of ouabainlike immunoreactive compound (50). Pharmacological blockade of brain ouabainlike factor with digoxin antibody in rat prevents the sympathetic hyperreactivity associated with high dietary NaCl intake and with air-jet stress (23, 24). Our present finding, that isolation stress alone markedly potentiates OLC, is consistent with those prior observations.

In the present study, natriuretic and diuretic responses occurring in both groups on high NaCl intake paralleled the gradual increases in MBG excretion. In the Iso group, increases in urinary excretion of sodium and potassium were associated with increases in OLC excretion. These observations are in agreement with previous observations of natriuretic and kaliuretic effects of MBG (3), ouabain (35), and OLC (26). Plasma levels of MBG and OLC positively correlated with renal excretion of these SPL on the final day of the experiment, suggesting that urinary SPL excretion may reflect their circulating levels throughout the experiment. However, further work is needed to substantiate this relationship.

The present study also demonstrated that high NaCl intake alone increases weights of the heart and kidney, whereas isolation stress alone increased weight of the aorta in the absence of increases in SBP. Previous studies have shown that a high-NaCl diet induced left ventricular (27, 33, 36) and renal (33, 36) hypertrophy in normotensive rats in the absence of a blood pressure increase. These increases in cardiac and kidney weight were not dependent on changes in plasma volume (36) and were insensitive to enalapril treatment (33). A novel finding of the present study is that, in response to increased dietary NaCl, the relative weights of hearts and kidneys were correlated with the magnitude of changes in the excretion of MBG but not with SBP or OLC. In contrast, the peak change in OLC but not MBG correlated with aortic weight. The latter is consistent with the recent finding in normotensive rats that acute restraint stress leads to hypertrophy of aorta rather than of myocardium (47). In our study, isolation stress alone, in the absence of an increase in SBP, was accompanied by significant increases in aortic weight, consistent with the previous finding that, after 35 days of intermittent isolation, the aortae of stressed rats were hypertrophied, and the hypertrophy was more pronounced in NaCl-loaded, isolated rats (8).

In conclusion, in normotensive F344 × NB rats, high NaCl intake per se appears to stimulate only MBG, whereas OLC is responsive to isolation stress. The combination of high NaCl intake and isolation stress is accompanied by marked stimulation of both OLC and MBG, which exert prohypertensive effects.

Perspectives

In fact, an increasing body of evidence implicates the involvement of SPL in the regulation of tissue growth. First, on the presumption of steroidal nature of digitalis-like factors, Schreiber and Kolbel (39) hypothesized that SPL might be involved in the regulation of tissue growth and myocardial hypertrophy. Subsequently, SPL, including bufadienolides, were shown to exert growth-promoting effects in different cell lines (34, 43), and an Na-K-ATPase inhibitory effect of ouabain was shown to initiate hypertrophic signaling in neonatal cardiac myocytes (25). More recently, in patients with essential hypertension, plasma levels of OLC were shown to independently predict increases in left ventricular mass (32), and elevated plasma and myocardial levels of digitalis-like immunoreactive material were associated with the development of hypertrophic cardiomyopathy (21). Thus regulation of growth may become a new feature of digitalis-like SPL.