Dietary Sodium Intake Modulates Vasodilation Mediated by Nitroprusside But Not by Methacholine in the Human Forearm
Abstract Studies in animals suggest that nitric oxide production is increased under conditions of salt loading and that this increase protects against the development of salt-induced hypertension. To determine the effect of dietary sodium intake on nitric oxide–mediated vascular responses, we studied seven healthy male volunteers twice 4 weeks apart while they were receiving a diet containing 10 or 250 mmol Na+ per 24 hours. Methacholine (0.25 to 8 μg/min) and sodium nitroprusside (0.25 to 8 μg/min) were infused intra-arterially in incremental doses, and forearm blood flow was measured. The response of forearm blood flow to sodium nitroprusside was greater when subjects received a high sodium diet than when they received a low sodium diet (F=7.11, P<.05); however, the response to methacholine was not altered by sodium intake (F=0.57, P=NS). Plasma renin activity was significantly higher (3.99 versus 1.0 ng angiotensin I/mL per hour) when subjects received a low salt diet (P<.05). Systolic pressure, diastolic pressure, heart rate, and baseline forearm blood flow were not affected by sodium status. We conclude that under conditions of salt loading, vasodilation in response to sodium nitroprusside was enhanced, whereas the response to methacholine was not affected, suggesting a differential effect of sodium intake on endothelium-dependent and endothelium-independent responses after the administration of methacholine and sodium nitroprusside, respectively.
Several vasodilators and vasoconstrictors produced and released by the endothelium regulate vascular tone.1 2 Nitric oxide (NO), an important endothelium-derived relaxing factor,3 is synthesized from l-arginine in a process that can be competitively antagonized by arginine analogues.4 The homeostatic function of NO-mediated vasodilation in the maintenance of vascular tone has been demonstrated in healthy subjects,5 and impaired endothelium-dependent vasodilation has been found in patients with hypertension,6 7 heart failure,8 and hypercholesterolemia,9 10 suggesting an important role for endothelium-mediated regulation of vascular tone in health and disease.
Several studies in animals indicate that NO plays a role in the regulation of the blood pressure response to sodium loading. In Sprague-Dawley rats, a high salt intake enhanced NO synthesis, as determined by the urinary excretion of nitrates and nitrites,11 and in dogs receiving a high sodium diet, inhibition of NO synthesis resulted in elevated arterial pressure and attenuation of both natriuresis and diuresis.12 When salt-sensitive and salt-resistant Dahl/Rapp rats received a high salt diet, the salt-sensitive strain produced less NO than the salt-resistant strain and became hypertensive, whereas the salt-resistant strain remained normotensive. However, l-arginine administration to the salt-sensitive strain, under similar conditions of salt loading, enhanced NO production to a level similar to that of the salt-resistant strain and prevented the development of hypertension.13 These studies suggest that salt loading in normotensive animals results in enhanced NO production and may alter NO-mediated responses, regulate natriuresis, and protect against the development of hypertension. Furthermore, in salt-sensitive strains, deficient NO production under conditions of salt loading may result in the development of hypertension. The relevance of these findings to humans is unclear; therefore, in this study we determined whether NO-mediated vasodilation was altered by sodium intake in normotensive subjects by examining forearm blood flow (FBF) responses to the endothelium-dependent agonist methacholine and the endothelium-independent agonist sodium nitroprusside.
Seven healthy, nonsmoking male volunteers aged 33.7±2.7 years were studied. No clinically significant abnormalities were found by history, physical examination, or routine laboratory tests, including complete blood count, prothrombin and partial thromboplastin times, renal and liver function tests, and electrocardiogram. Subjects did not take any medications for at least 2 weeks before each study day and abstained from caffeine and alcohol for 5 days before each study day. All subjects provided written informed consent; the study protocol was approved by the Vanderbilt Committee for the Protection of Human Subjects.
Subjects were studied on two occasions, 4 weeks apart, with identical procedures followed on each study day. For 5 days before each study day, subjects were maintained on a diet containing either 10 or 250 mmol Na+ per 24 hours. The diet was prepared by the metabolic kitchen of the Vanderbilt Clinical Research Center under the supervision of a dietitian. The order of administration of the low or high sodium diet was randomized. To demonstrate that sodium balance had been attained, 24-hour urinary sodium excretion was determined in samples collected on the fifth day of both low and high sodium diets. All experiments were performed in the morning, in the same temperature-controlled room, by the same investigators, with the subjects resting supine in bed. An intravenous cannula was placed in an antecubital vein of both arms. After subdermal administration of 1% lidocaine, an 18-gauge polyurethane catheter (Cook Inc) was inserted into the brachial artery of the nondominant arm, allowing direct intra-arterial drug administration. Arterial catheter patency was maintained with an infusion of 40 mL/h of 5% dextrose in water. During intra-arterial drug administration, the total flow rate through the cannula was maintained constant at 40 mL/h by altering the concentration of the drug infusion. Arterial blood pressure was measured by means of a pressure transducer (Hewlett-Packard), and heart rate was recorded from a continuous electrocardiograph monitor. After the arterial line and intravenous catheters had been placed, subjects rested quietly for 30 minutes before baseline measurements were determined. Venous blood samples for renin determination were collected in chilled tubes containing EDTA, centrifuged at 4°C, separated immediately, and stored at −20°C until analyzed. Arterial blood samples for determination of norepinephrine concentrations were collected in cooled tubes with EGTA and reduced glutathione (Amersham Corp), placed on ice, centrifuged at 4°C, stored, and later assayed in duplicate, as described below.
Methacholine and sodium nitroprusside were infused intra-arterially in incremental doses (0.25 to 8 μg/min) with an infusion pump (Harvard Apparatus). Each dose was infused for 5 minutes, with FBF measurements made during the last 2 minutes of each infusion. The order of methacholine and sodium nitroprusside administration was randomized between subjects, but each subject received these two drugs in the same order on the 2 study days. A 30-minute washout period between the infusion of the two drugs allowed blood flow to return to baseline before infusion of the second drug.
Forearm Blood Flow
FBF was measured in the arm into which intra-arterial vasodilators were infused with the use of mercury-in-Silastic strain-gauge plethysmography.14 The wrist was supported in a sling so the level of the forearm was raised above that of the atrium. The hand was excluded from the measurement of blood flow by inflation of a pediatric sphygmomanometer cuff to 200 mm Hg around the wrist before and during FBF measurement. Forearm volume, excluding the hand and wrist, was measured by water displacement. Blood flow tracings were analyzed manually by a single investigator who was blinded to sodium intake on the different study days.
Plasma renin activity was determined by generation of angiotensin I (Ang I) measured by radioimmunoassay.15 Norepinephrine concentrations were measured by high-performance liquid chromatography with electrochemical detection and 3,4-dihydroxybenzylamine as the internal standard as described previously16 17 ; urinary sodium concentrations were measured by flame photometer.
Data are expressed as mean±SEM and were analyzed by repeated-measures ANOVA or Student’s t test for paired data as appropriate. A value of P<.05 was accepted as the minimal level of significance.
The Table⇓ shows baseline characteristics of the seven subjects under conditions of sodium depletion and sodium loading. Sodium intake did not alter systolic pressure, diastolic pressure, or heart rate. Twenty-four-hour urinary sodium excretion demonstrated good compliance with the diet, and concomitantly, plasma renin activity was significantly higher (3.99±0.92 versus 1.0±0.1 ng Ang I/mL per hour) when subjects received a low sodium diet (P<.05). Baseline FBF on the low sodium study day (2.78±0.3 mL/100 mL per minute) did not differ from that on the high sodium study day (3.45±0.5 mL/100 mL per minute, P=NS). The concentration of arterial plasma norepinephrine was significantly higher when subjects received the low sodium diet (179.8±20.2 versus 120.9±20.2 pg/mL, P<.005). Neither plasma norepinephrine concentrations nor plasma renin activity correlated with FBF responses to 8 μg/min methacholine or sodium nitroprusside during either the low or high sodium diet.
Mean FBF responses to methacholine (Fig 1⇓) were not altered by sodium intake (F=0.57, P=.48); however, responses to sodium nitroprusside (Fig 2⇓) were enhanced when subjects received a high sodium diet (F=7.11, P<.05).
In this study we found that the forearm vascular response to methacholine was not influenced by dietary salt intake; however, responses to sodium nitroprusside were significantly greater when subjects were receiving a high salt diet.
Several studies in animals suggest that dietary sodium intake may modulate NO production. Exposure of rats to a high salt intake resulted in increased concentrations of plasma and urinary nitrates and nitrites, products of NO metabolism,11 and compared with controls, the chronically salt-loaded rats demonstrated enhanced renal hemodynamic responses to inhibition of NO synthase,11 suggesting that the endogenous NO system modulates renal hemodynamics and participates in the physiological adaptation to increased salt intake. The possibility that impaired NO synthesis contributed to the pathogenesis of salt-sensitive hypertension was suggested by studies demonstrating that increased dietary salt intake resulted in increased NO production in salt-resistant but not salt-sensitive rats and that administration of l-arginine, the precursor of NO, resulted in increased NO production and prevention of hypertension in salt-sensitive rats.13 18 These studies therefore suggested that the normal response to salt loading was an increase in NO production and that salt-sensitive hypertension developed in rat strains in which this response was impaired.
Sodium intake has been found to influence NO-mediated vascular responses in addition to modulation of NO production. Boegehold19 found that high salt intake resulted in impaired vasodilation in response to acetylcholine in the arcade arterioles but not in the smaller transverse and distal arterioles in the spinotrapezius muscle microvasculature of salt-sensitive but not salt-resistant Dahl rats. Responses to sodium nitroprusside were not altered, suggesting that the effect of salt was not due to altered smooth muscle responsiveness to NO.19
In the present study sodium intake did not influence the methacholine-induced increase in FBF. Acetylcholine and methacholine, endothelium-dependent vasodilators, are thought to act on endothelial muscarinic receptors, resulting in the activation of NO synthase, which stimulates NO production from l-arginine.1 20 In contrast, sodium nitroprusside, an endothelium-independent vasodilator, is thought to release NO directly through poorly understood mechanisms.1 20 The demonstration of membrane-associated, sodium nitroprusside–directed, NO-generating activity in bovine coronary artery smooth muscle subcellular fractions21 and of NO release from sodium nitroprusside after reaction with thiols, reducing agents, hemoglobin, myoglobin, and partially purified cytochrome P-45022 suggests that the production of NO from sodium nitroprusside in biological tissue is complex and not caused by spontaneous degradation, as was previously thought. Therefore, although both methacholine and sodium nitroprusside activate soluble guanylate cyclase through NO to produce smooth muscle relaxation and vasodilation, they produce NO by different mechanisms.
We used sodium nitroprusside and methacholine in the present study so that we could define the site of any abnormality in NO responsiveness. The finding of increased sensitivity to sodium nitroprusside during high sodium intake, in the face of an unaltered response to methacholine, would not exclude the possibility that sodium loading might result in increased vascular reactivity to NO (as shown by the sodium nitroprusside response) if the unaltered response to methacholine could be accounted for by an inhibitory effect of high sodium intake on endothelium-dependent NO synthesis after methacholine administration. An alternative interpretation is that the vascular response to NO was unaltered by sodium status and that the enhanced response observed after sodium nitroprusside administration while subjects received a high sodium diet was due to enhanced endothelium-independent generation of NO from sodium nitroprusside during conditions of high salt intake.
It is difficult to exclude the possibility of a sodium-induced alteration in drug clearance, or at least local drug clearance in the forearm. However, the strength of the technique of agonist delivery directly into the arterial system under investigation is that it provides the advantage of the delivery of an exact drug dose into the vascular bed whose response is being determined; for this reason the technique has been used extensively for the study of the regulation of vascular tone.6 7 8 9 10 However, it is possible that sodium intake may alter local NO degradation. Another possibility is that the interday variability in the methacholine responses was greater than that of the sodium nitroprusside response, obscuring an effect of sodium intake on the response to methacholine. We do not have data on the interday variability of methacholine or nitroprusside responses in individuals studied twice while on the same sodium intake, but the variance for methacholine and sodium nitroprusside responses in the present study was similar, suggesting that greater variability in the responses to methacholine is unlikely to account for our findings.
Modulation of FBF by altered sodium intake has been observed when isoproterenol, a drug that stimulates adenylate cyclase through β-receptors, was used to examine the effects of sodium intake on β-receptor–mediated responses.23 Naslund and colleagues23 found that in normotensive but not hypertensive subjects, a high sodium intake resulted in increased sensitivity to isoproterenol-induced vasodilation and that this was accompanied by a parallel increase in the proportion of β-receptors exhibiting binding in the high-affinity state, suggesting that the observed alterations in responsiveness to isoproterenol were accounted for by alterations in the β-receptor. However, since the generation of NO from sodium nitroprusside is not thought to be receptor mediated, it is unlikely that mechanisms similar to those thought to account for sodium-induced alterations in β-receptor responsiveness explain the enhanced response to sodium nitroprusside. However, it is possible that a high sodium intake through a mechanism as yet undetermined results in a generalized enhanced vascular smooth muscle responsiveness to endothelium-independent agonists. Therefore, further studies examining the effects of sodium intake on other endothelium-independent agonists would be of interest. Studies examining the effects of sodium intake on responses to bradykinin and the reactive hyperemia response to ischemia, stimuli acting partially through NO, would also be of interest.
In conclusion, the administration of a high salt diet to normotensive subjects did not alter NO-mediated vascular responsiveness as determined by methacholine-induced increases in FBF; however, responses to sodium nitroprusside were enhanced for reasons that are at present unclear.
This work was supported in part by a Grant-in-Aid from the American Heart Association, Tennessee Affiliate, and US Public Health Service grants GM 31304, GM 46622, and GM 5M01-RR00095.
Reprint requests to Dr Alastair J.J. Wood, Division of Clinical Pharmacology, Vanderbilt University School of Medicine, Medical Research Building, Room 546, Nashville, TN 37232-6602.
- Received September 7, 1994.
- Revision received October 25, 1994.
- Accepted January 18, 1995.
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