The endothelium-dependent forearm vasodilation

Transcription

1 AJH 1997;10: ORIGINAL COMMUNICATIONS Impairment of Endothelial Function in Salt-Sensitive Hypertension in Humans Aya Miyoshi, Hiroshi Suzuki, Masayoshi Fujiwara, Miho Masai, and Tadaaki Iwasaki In this study, we evaluated the relationship between the endothelium-dependent vasodilation and salt sensitivity in patients with essential hypertension. Fifteen untreated hypertensive male patients (age, 29 to 54 years) were sodium restricted (5 g/day) for 1 week, and placed on a high salt diet (20 g/day) the second week. At the end of each period, measurements of forearm vascular responses to drugs (acetylcholine, 3 to 24 g/min; sodium nitroprusside, 0.15 to 1.2 g/min; norepinephrine, 0.15 to 1.2 g/min; and N G - monomethyl-l-arginine [L-NMMA], 1 to 8 mol/ min) were obtained by using strain-gauge venous plethysmography. Subjects were divided into two groups according to the blood pressure response to sodium loading: salt-sensitive hypertensive group (24-h mean increase of arterial pressure >10%; n 6) and salt-resistant group (<10%; n 9). The two groups showed no significant difference in clinical The endothelium-dependent forearm vasodilation in response to acetylcholine is reduced in patients with essential hypertension. 1 4 A factor released by the endothelial cells determines vascular tone. The so-called endothelium-derived relaxing factor has been identified as nitric oxide (EDNO). 5 7 Chen and Sanders 8 observed that the prolonged administration of l-arginine, a substrate for nitric oxide synthesis, prevents the development of Received April 1, Accepted April 7, From The First Department of Internal Medicine, Hyogo College of Medicine, Nishinomiya, Japan. Address correspondence and reprint requests to Aya Miyoshi, The First Department of Internal Medicine, Hyogo College of Medicine, 1-1 Mukogawa-cho, Nishinomiya, 663, Japan. data or mean arterial pressure during low salt intake. The dose-dependent vasodilation induced by acetylcholine was significantly reduced (P <.05) in the salt-sensitive hypertensive patients v the salt-resistant patients regardless of sodium loading. There were no differences between the two groups in response to sodium nitroprusside, norepinephrine, or L-NMMA. These results indicate that vasodilation to acetylcholine is reduced in salt-sensitive hypertensive patients even on restricted sodium diets. This may contribute to blood pressure elevation when sodium intake is increased. Am J Hypertens 1997; 10: American Journal of Hypertension, Ltd. KEY WORDS: Salt-sensitive hypertensive patients, acetylcholine, strain-gauge venous plethysmography. hypertension and increases urinary cyclic GMP in Dahl salt-sensitive rats exposed to a high salt diet. Other investigators report that NO synthase stimulation in vivo induces natriuresis and diuresis These findings suggest that the activity of the NO system, especially in renal sodium handling, may be related to salt sensitivity in rats. Despite this information, the relation between salt sensitivity and endothelial function in systemic resistance vessels of hypertensive humans is still unknown. We conducted this study to determine whether the endothelium-dependent vasodilation in response to acetylcholine differs in patients with salt-sensitive and salt-resistant essential hypertension. If so, we will attempt to determine whether such differences are attributable to a dysfunction in the nitric oxide system by the American Journal of Hypertension, Ltd /97/$17.00 Published by Elsevier Science, Inc. PII S (97)

2 1084 MIYOSHI ET AL AJH OCTOBER 1997 VOL. 10, NO. 10, PART 1 METHODS Subjects We studied 15 untreated hypertensive patients aged years (range, 29 to 54 years) (Japanese men). Antihypertensive medications were withheld for at least 4 weeks before the start of the study. After 5 min of rest, sitting blood pressure was measured by a physician using a mercury sphygmomanometer on at least three different occasions in the outpatient clinic. Patients were considered hypertensive if their systolic or diastolic blood pressure was consistently 140 mm Hg or 90 mm Hg, or both, respectively. Patients were admitted throughout the study and blood samples were obtained for blood cell counts, serum electrolytes, creatinine, fasting blood sugar, cholesterol, triglycerides, liver enzymes, plasma renin activity, aldosterone, and catecholamine measurements. Chest x-rays, electrocardiograms, echocardiograms, and funduscopic examination were taken. All patients were either stage I or II hypertensive as defined by the World Health Organization classification. Secondary forms of hypertension were excluded on routine examination. Patients with diabetes mellitus and hyperlipidemia and alcoholism were excluded from the study. Some of these patients were smokers and habitual drinkers, but they stopped this activity before the experiment. Drugs All drugs were obtained from commercially available sources. Acetylcholine HCl (Daiichi Seiyaku, Tokyo, Japan) and N G -monomethyl-l-arginine (L- NMMA; Sigma Chemical Company, St. Louis, MO) were lyophilized and dissolved (strength of acetylcholine, 12 g/ml; l-nmma, 4.0 mol/ml) in physiologic saline immediately before use. Sodium nitroprusside (Wako Junyaku Kogyo, Osaka, Japan) and norepinephrine (Sankyo Corp., Tokyo, Japan) were dissolved (strength of sodium nitroprusside, 0.6 g/ ml; norepinephrine, 20 ng/ml) in physiologic saline. All solutions were passed through a bacterial filter (Millipore Limited, Tokyo, Japan) immediately before use. Sodium nitroprusside was protected from light. Protocol The protocol was explained, and written informed consent for participation was obtained from each subject. In the first week of the experiment, each subject received a low sodium diet (5 g/day), and in the second week, coated tablet of sodium chloride (Wako Junyaku Kogyo; 500 mg per tablet) was added to this diet to achieve a high sodium intake (20 g/day). A glucose tolerance test (75 g oral glucose) was administered to evaluate glucose tolerance in the end of the sodium-restricted period. The sum of blood sugar divided by the sum of immune reactive insulin ( BS/ IRI) was then calculated to evaluate insulin resistance. Immune reactive insulin was measured by standard laboratory practice. At the end of each week, measurements of forearm vascular responses to drugs along with 24-h ambulatory blood pressures were monitored (ABPM 630; Nippon Colin Co. Ltd., Komaki, Japan). Ambulatory blood pressure was measured every 30 min between 8 am and 8 pm, and every hour between 8 pm and 8 am. An average of the mean arterial pressure (MAP) over the 24 h was calculated. The hypertensive subjects were divided into two groups: salt sensitive (SS; increase of MAP 10%) and salt resistant (SR; increase of MAP 10%). Forearm Vascular Response to Drugs All studies were started at 9 am after an overnight fast with the subject lying supine in a quiet air-conditioned room (22 to 24 C). Under local anesthesia (2% lidocaine), a 20-gauge Teflon cannula (Angiocath; Becton Dickinson Vascular Access, UT) was inserted into the left brachial artery just below the elbow joint, where the pulsation of the brachial artery was well palpable. Each study consisted of the infusion of drugs into the brachial artery followed by the measurement of the forearm vascular tone by strain-gauge venous occluded plethysmography. The cannulated arm was slightly elevated and the strain gauge was fitted on to the widest part of the forearm. 13,14 The strain gauge was connected to a plethysmograph (Medasonics, Fremont, CA) and a chart recorder. For each measurement, a cuff placed on the wrist was inflated to suprasystolic blood pressures, 15 and a cuff placed on the upper arm was immediately inflated to 40 mm Hg. Blood flow in the forearm was measured five times for each dose and was expressed as milliliters per minute per 100-milliliters of forearm tissue according to the method of Whitney. 16 Arterial blood pressure was recorded directly from the intraarterial catheter immediately before each measurement. Vascular resistance in the forearm was calculated as the mean pressure divided by the blood flow in the forearm. To rule out the influences of the precontracted level for the vasoreaction for drugs, correlation between basal and the changes of forearm vascular resistance was examined according to the method by Overbeck et al. 17 Endothelium-dependent vasodilation was assessed by measuring the response to an intraarterial infusion of acetylcholine (infused at 3, 6, 12, and 24 g/min). Endothelium-independent vasodilation was estimated with an intraarterial infusion of sodium nitroprusside (infused at 0.15, 0.3, 0.6, and 1.2 g/min). Vasoconstrictor responses were estimated by the infusion of norepinephrine (infused at 0.15, 0.3, 0.6, and 1.2 g/ min), and of L-NMMA (infused at 1, 2, 4, and 8 mol/ min), a stereospecific inhibitor of nitric oxide synthesis. Infusion rates were 0.25, 0.5, 1, and 2 ml/min, respectively, for each drug. Each dose was infused for 8 min (with forearm blood flow measured during the final 3 min). Thirty minutes of recovery was allowed

3 AJH OCTOBER 1997 VOL. 10, NO. 10, PART 1 SALT-SENSITIVE HTN AND ACH VASODILATION 1085 TABLE 1. CHARACTERISTICS OF THE STUDY POPULATION Characteristics SR (n 9) SS (n 6) Age (years) Height (cm) Weight (kg) Cardiothoracic ratio (%) Serum sodium (meq/l) Creatinine (mg/dl) Total cholesterol (mg/dl) Triglycerides (mg/dl) Fasting blood sugar (mg/dl) of blood sugar/ of immune reactive insulin Habitual drinker 4/9 3/6 Smoker 5/9 3/6 Data are expressed as mean SE. All data reflect nonsignificant differences between the two groups. SR, salt-resistant group; SS, salt-sensitive group. between the four experimental steps and we validated that forearm blood flow and vascular resistance were normalized. Statistical Analysis Results are expressed as mean SE. The statistical significance of the differences between two means was assessed by paired or unpaired Student s t tests, as appropriate. The responses to drugs were compared by ANOVA for repeated measures. Correlation of the data was determined with a least-squares fit analysis. A level of P.05 was accepted as statistically significant. RESULTS Salt Sensitivity of Hypertensive Patients There were six patients who were defined as SS, and nine as SR. There were no significant differences in clinical data during low salt intake and in mean blood pressure in the outpatient clinic under usual diet between SS and SR (Tables 1 and 2). Left ventricular hypertrophy was not observed in any patients using electrocardiogram and echocardiogram. Aterioscopic retinopathy was observed in one patient, who was diagnosed as SR. One SS patient was diagnosed as having glucose intolerance, but there was no difference in the BS/ IRI between the two groups. Although lower plasma renin activity was observed in the SS group, it was not significant. There was no significant difference in the average of mean arterial pressure during low sodium diet between SR and SS. During high salt intake, there was an increase in mean arterial pressure in SS, an increase in urine volume and urinary sodium excretion, a reduction in plasma renin activity, and a lower hematocrit in each group (Table 2). Responses to Acetylcholine Basal forearm blood flow and vascular resistance were not different between the two groups during both low and high sodium intake (Table 2). The intraarterial infusion of acetylcholine induced a dose-dependent vasodilation in all subjects. During both low and high sodium diets, TABLE 2. CHANGES IN CLINICAL PARAMETERS DURING LOW AND HIGH SODIUM INTAKE Parameters SR Low Salt High Salt Low Salt High Salt Average of mean arterial pressures (mm Hg) * Urine volume (ml/day) * * Urinary sodium excretion (meq/day) Hematocrit (%) * * Plasma renin activity (ng/ml/h) * * Serum aldosterone (pg/ml) Urinary epinephrine excretion ( g/day) Urinary norepinephrine excretion ( g/day) Basal forearm blood flow (ml/100 ml/min) Total peripheral resistance (mm Hg/mL/100 ml/min) SS Data are expressed as mean SE. *P.05; P.01 v low salt diet. SR salt-resistant group; SS salt-sensitive group.

4 1086 MIYOSHI ET AL AJH OCTOBER 1997 VOL. 10, NO. 10, PART 1 FIGURE 1. (A) Forearm blood flow and vascular resistance responses to acetylcholine during low sodium intake in nine salt-resistant hypertensive patients (open circles) and six salt-sensitive patients (closed circles). Data are expressed as mean SEM. (B) Forearm blood flow and vascular resistance responses to acetylcholine during high sodium intake in nine salt-resistant hypertensive patients (open circles) and six salt-sensitive patients (closed circles). Data are expressed as mean SEM. *P.05, **P.01 v low sodium intake. response of forearm blood flow to acetylcholine were significantly reduced in SS compared with SR (P.05) (Figure 1A upper panel and B upper panel). In addition, vascular resistance caused by acetylcholine was significantly lower in SS compared with SR (P.05) (Figure 1A bottom panel and B bottom panel), and the correlation between basal and the change of forearm vascular resistance was different in SR and SS (Figure 2). After 1 week of high sodium intake, each group exhibited a slight increase in blood flow and a decrease in forearm vascular resistance at the highest acetylcholine dose, but it was significant in only SS [percent forearm blood flow in SS at the dose of 24 g/min was in the low sodium taking period and in the high sodium taking period (P.05) and percent forearm vascular resistance in SS at the dose of 24 g/min was in the low sodium taking period and in the high sodium taking period (P.01)] (Figure 1B). Responses to Sodium Nitroprusside and Norepinephrine The infusion of sodium nitroprusside produced dose-dependent vasodilation, whereas norepinephrine induced dose-dependent vasoconstriction in all patients. There were no significant differences between the responses in the SS and SR groups, and sodium loading had no effect on either group (Figures 3 and 4). No differences were observed between each group in the response of forearm vascular resistance and the correlation between basal and the change in forearm vascular resistance (data not shown). Responses to N G -Monomethyl-L-arginine L-NMMA produced a dose-dependent decrease in the forearm blood flow of all subjects. There was no difference between the forearm blood flow response of the SS and SR groups before or after sodium loading (Figure 5). No differences were observed between groups in the response of forearm vascular resistance and the

5 AJH OCTOBER 1997 VOL. 10, NO. 10, PART 1 SALT-SENSITIVE HTN AND ACH VASODILATION 1087 FIGURE 2. Scatterplot and regression lines showing correlation between basal forearm vascular resistance and the change in forearm vascular resistance produced by acetylcholine infusion at 24 mol/min in nine salt-resistant hypertensive patients (open circles) and six saltsensitive patients (closed circles). 95% confidence intervals for predicted values were also drawn. correlation between basal and the change in forearm vascular resistance (data not shown). Infusion of each drug did not induce any changes in blood pressure or heart rate and forearm blood flow in the noninfused arm, confirming that these drugs did not have systemic effect at the doses used. DISCUSSION The pathogenesis of essential hypertension is multifactorial, with sodium retention being one of the contributing factors, 18 hypothetically causing an increase in intercellular sodium and calcium. 19 Salt-sensitive patients with hypertension show a greater tendency to retain sodium, 20 an increase or insufficient suppression in plasma catecholamine levels, a decreased urinary excretion of dopamine, 24 a lower kallikreinkinin activity, and impaired glucose tolerance. 28,29 In addition to these abnormalities, the present study demonstrated that the vasodilation induced by acetylcholine was significantly lower in salt-sensitive versus salt-resistant hypertensive patients regardless of sodium loading. Conversely, these groups showed comparable responses to sodium nitroprusside and norepinephrine. Previous studies have demonstrated that the vasodilatory response to the intraarterial administration of acetylcholine is blunted in patients with essential hypertension. 1 4 This altered vascular response is considered to result from a dysfunctional vascular endothelium. 2,30,31 This holds true because acetylcholine induces endothelium-dependent vascular dilation by releasing vasorelaxing substances. Because some studies reported that antihypertensive treatment restored this impaired endothelium-dependent vascular relaxation in salt-induced hypertensive rats, 32 we have to consider the possibility that reduced vasodilation in response to acetylcholine in salt-sensitive patients in our study may be the result of high blood pressure. On the other hand, other studies reported that clinically effective antihypertensive therapy does not restore the reduced response to acetylcholine in patients with essential hypertension 33 and a decreased vasodilatory response to acetylcholine is also observed in the normotensive offspring of hypertensive patients. 34 Therefore, it is still unknown whether the endothelial dysfunction of hypertensive patients is a primary phenomenon or the result of high blood pressure. In our study, because we could not find any differences in blood pressure levels with a normal or sodium-restricted diet and overall patient characteristics in either group, the altered response to acetylcholine in salt-sensitive patients seems unlikely the result of higher blood pressure of patients in salt-sensitive hypertension, but might be one of the predisposing factors for the increase in blood pressure with high sodium intake. Salt-sensitivity is common in specific patient populations, such as blacks, 35 the elderly, 36,37 obese people, and those with glucose intolerance. 28,29 Because our subjects were all Japanese men with no differences in age and body mass index between groups, a reduced acetylcholine responsiveness in salt-sensitive hypertensive patients could not be attributable to population differences. The BS/ IRI values were also similar in both groups. As described, one salt-sensitive hypertensive patient was diagnosed as being glucose intolerant, but the acetylcholine response of that patient did not differ from that of the other salt-sensitive

6 1088 MIYOSHI ET AL AJH OCTOBER 1997 VOL. 10, NO. 10, PART 1 FIGURE 3. Forearm blood flow responses to sodium nitroprusside in nine salt-resistant hypertensive patients (open circles) and six salt-sensitive patients (closed circles). The upper panel indicates low sodium responses and the lower panel indicates high sodium responses. Data are expressed as mean SEM. patients. Further investigation of insulin resistance using the glucose clamp test would clarify this issue. A high sodium load slightly increased the vasodilator response to acetylcholine in each group, but it was statistically significant only in salt-sensitive patients. Our results suggest that sodium overloading itself does not cause impaired vasodilation in a short time period such as 1 week. However, as we did not investigate the influence of sodium overloading on drug vasoreactions in normal control subjects, it is not known whether sodium loading increases endothelial function in normotensive subjects. Vascular endothelial cells release several factors that act on vascular smooth muscle in the presence of acetylcholine. Such substances include endothelium-derived relaxing factor (EDRF or EDNO; nitric oxide), endothelium-derived hyperpolarizing factor (EDHF), 38 and endothelium-derived contracting factors (EDCF; endothelin and constrictor prostanoids). 39,40 To identify which of these agents is involved in the altered responsiveness to acetylcholine in salt-sensitive hypertensive patients, we evaluated the vasoreaction to L-NMMA, a nitric oxide synthase inhibitor. Previous studies report that vasoconstrictive responses to intraarterial L-NMMA were lower in hypertensive patients. 41 In our study, responses to L-NMMA were similar in both salt-sensitive and saltresistant patients. This contradicts the report of Chen and Sanders, 8 which attributed the decreased EDNO levels to a deficiency in substrate for nitric oxide synthesis in Dahl salt-sensitive rats exposed to high salt intake. They monitored endogenous EDNO production by measuring urinary excretion of cyclic GMP. It is possible that urinary excretion of cyclic GMP may not completely reflect the endothelial functions in systemic resistance vessels. Also, the pathogenesis of Dahl salt-sensitive hypertension in rats may differ from that of human saltsensitive hypertension. According to our results, it is likely that the basal release of nitric oxide was the same in the salt-sensitive and salt-resistant groups, and suggests that basal EDNO does not participate in a salt- FIGURE 4. Forearm blood flow responses to norepinephrine in nine salt-resistant hypertensive patients (open circles) and six salt-sensitive patients (closed circles). The upper panel indicates low sodium responses and the lower panel indicates high sodium responses. Data are expressed as mean SEM.

7 AJH OCTOBER 1997 VOL. 10, NO. 10, PART 1 SALT-SENSITIVE HTN AND ACH VASODILATION 1089 FIGURE 5. Forearm blood flow responses to N G -monomethyll-arginine in nine salt-resistant hypertensive patients (open circles) and six salt-sensitive patients (closed circles). The upper panel indicates low sodium responses and the lower panel indicates high sodium responses. Data are expressed as mean SEM. induced increase in blood pressure. Therefore, decreased EDHF or increased EDCF is considered to be responsible for the attenuated vascular response in salt-sensitive patients. The impaired endothelial vasodilation has been reported to be attributable to increased vasoconstrictor prostaglandin in spontaneously hypertensive rats. 42 But there exists the possibility that an acetylcholine-mediated pathway is selectively impaired in endothelial nitric oxide release. Because we have not investigated normotensive controls, it is difficult to determine that the response to L-NMMA was altered in either salt-sensitive or salt-resistant hypertensive patients. Using the acetylcholine test as an evaluation of endothelium function may cause problems as the change in the endothelial or smooth muscle cell muscarinic receptor population is not necessarily associated with a decrease in nitric oxide release. 43 In summary, we observed a decreased response to acetylcholine in salt-sensitive hypertensive patients. This may not be attributable to an altered basal EDNO release because there is no difference in response to L-NMMA. Further investigation is required to clarify the mechanism of these impaired acetylcholine responses. REFERENCES 1. Panza JA, Quyyumi AA, Brush JE, Jr., et al: Abnormal endothelium-dependent vascular relaxation in patients with essential hypertension. N Engl J Med 1990;323: Linder L, Kiowski W, Buhler FR, et al: Indirect evidence for release of endothelium-derived relaxing factor in human forearm circulation in vivo: blunted response in essential hypertension. Circulation 1990;81: Panza JA, Casino PR, Badar DM, et al: Effect of increased availability of endothelium-derived nitric oxide precursor on endothelium-dependent vascular relaxation in normal subjects and in patients with essential hypertension. Circulation 1993;87: Panza JA, Casino PR, Kilcoyne CM, et al: Role of endothelium-derived nitric oxide in the abnormal endothelium-dependent vascular relaxation of patients with essential hypertension. Circulation 1993;87: Furchgott RF, Zawadski JV: The obligatory role of endothelial cells in the relaxation of arterial smooth muscle by acetylcholine. Nature 1980;288: Furchgott RF: Role of endothelium in response of vascular smooth muscle. Circ Res 1983;53: Palmer RMJ, Feggige AG, Moncada S: Nitric oxide release accounts for the biological activity of endothelium-derived relaxing factor. Nature 1987;327: Chen PY, Sanders PW: l-arginine abrogates salt-sensitive hypertension in Dahl/Rapp rats. J Clin Invest 1991; 88: Zatz R, DeNicci G: Effects of acute nitric oxide inhibition on rat glomerular microcirculation. Am J Physiol 1991;261: Baylis C, Harton P, Engels K: Endothelial derived relaxing factor controls renal hemodynamics in the normal rat kidney. J Am Soc Nephrol 1990;1: Shultz PS, Tolins JP: Adaptation to increased dietary salt intake in the rat: role of endogenous nitric oxide. J Clin Invest 1993;91: Patel A, Layne S, Watts D, et al: l-arginine administration normalizes pressure natriuresis in hypertensive Dahl rats. Hypertension 1993;22: Whitney RJ: Measurement of changes in human limb volume by means of a mercury-in-rubber strain gauge. J Physiol 1948;109: Greenfield ADM, Whitney RF, Mowbray JF: Methods for the investigation of peripheral blood flow. Br Med Bull 1963;19: Kerslake DM: The effect of the application of an arterial occlusion cuff to the wrist on the blood flow in the human forearm. J Physiol (Lond) 1949;108: Whitney RJ: The measurement of volume changes in human limbs. J Physiol (Lond) 1953;121:1 27.

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