Weight Reduction Decreases the Circulating Concentration of the N-terminus of the AN F Prohormone ROBERT

w. McMURRAY, JR, MD,* DAVID L. VESELY, MD, PHDt

ABSTRACT: The N-terminus of the atrial natriuretic factor (ANF) prohormone (ie, proANF 1-98) contains two vasodilatory peptides consisting of amino acids (aa): aa 1-30 (ie, proANF 1-30) and aa 31-67 (ie, proANF 31-67) of the 126 aa prohormone. The relationship of this Nterminus to the renin-aldosterone axis and blood pressure reduction was investigated in 18 obese subjects (5 hypertensive and 13 normotensive) placed on a 12-week, low sodium (40 mmol), weight reducing diet. The N -terminus of the ANF prohormone and proANF 31-67, which circulates as a distinct entity after being proteolytically cleaved from the N-terminus, were significantly (p < 0.001) higher (767 ± 1.01 and 816 ± 135 fmol/ml) in the obese hypertensive group compared with the obese normotensive group (377 ± 24 and 356 ± 17 fmol/ml, respectively) prior to beginning the weight reduction program. There was a dramatic fall in the N -terminus and in proANF 31-67 after 1 week of weight reduction in both obese groups, which correlated with the decrease in mean arterial pressure during the first week and throughout the 12 weeks of weight reduction (r = .54, p < 0.001 and r = .59, p < 0.001, respectively). ProANF 1-98 had a significant (p < 0.01) inverse correlation with plasma renin in both obese From the t Departments of Medicine, Physiology, and Biophysics, University of South Florida Health Sciences Center, James A. Haley Veterans Hospital, .Tampa, Florida, and the *Department of Internal Medicine, University of Arkansas for Medical Sciences, John L. McClellan Memorial Veterans Hospital, Little Rock, Arkansas. Dr. McMurray's present address is Department of Internal Medicine, University of Missouri School of Medicine, One Hospital Drive, Columbia, MO 63212. This investigation was presented in part at the 73rd Annual Meeting of the Endocrine Society, June 19-22, 1991, in Washington, DC. This. investigation was supported in part by a Merit Review Grant from the United States Department of Veteran Affairs and by the Mr. & Mrs. Sam Walton Research Award to Dr. Vesely. The authors thank Amy Giordano for excellent secretarial help. Correspondence: David L. Vesely, MD, PhD, Professor of Medicine, James A. Haley Veterans Administration Medical Center-151, 13000 Bruce B. Downs Blvd., Tampa, FL 33612.

2

groups. ProANF 31-67, likewise, had an inverse correlation with plasma renin in the hypertensive (p < 0.002), as well as the normotensive (p < 0.03) subjects. ProANF 31-67 did not significantly correlate with aldosterone in either group. ProANF 1-98 had an inverse correlation with aldosterone only in the normotensive group (r = .33, p < 0.01), but not in the hypertensive group. These results demonstrate that weight loss while ingesting a controlled low sodium diet is accompanied by changes in the N -terminus of the ANF prohormone and proANF 31-67, which correlate with changes in blood pressure and inversely correlate with changes in plasma renin activity (PRA). ProANF 1-98 and proANF 3167 did not correlate with plasma aldosterone concentration in either group, as ANF has been shown to do previously. These findings reflect recent in vitro data that proANF 1-30 and proANF 31-67 have different mechanisms of action at the cellular level because they do not inhibit angiotensin II-stimulated aldosterone production while ANF does inhibit this secretion. KEY INDEXING TERMS: Atrial natriuretic factor; Hypertension; Obesity; Weight loss; Prohormone; Aldosterone; Renin. [Am J Med Sci 1992; 30~(1):2-8.1

A

trial natriuretic factor (ANF), the 28 amino acid (aa) carboxy-(C)-terminal end of the 126 aa ANF prohormone has natriuretic, diuretic, and potent vasodilator properties. 1,2 Atrial natriuretic factor has recently been found to decrease in the circulation with weight reduction and to correlate directly with the weight loss-induced decrease in blood pressure in both obese hypertensive and normotensive individuals.3 During the 12 weeks of a weight reduction diet, circulating ANF levels inversely correlated with changes in the rellin-aldosterone axis. 3 These observations are consistent #ith the ability of ANF to inhibit aldosterone secretion both in vitro and in vivo4- s and its ability to inhibit renin release. 7,8 January 1992 Volume 303 Number 1

McMurray and Vesely

In addition to ANF, there are at least two other peptides from the ANF prohormone with potent blood pressure lowering effects in vivo. 9 Two of these peptides from the N-terminus of the ANF prohormone, consisting of aa 1-30 (proANF 1-30; Long Acting Sodium Stimulator) and aa 31-67 (proANF 31-67; Vessel Dilator), also vasodilate vasculature in vitro.1O Since the whole N-terminus, consisting of aa 1-98, containing these vasodilatory peptides as well as the C-terminus (ANF, aa 99-126) of this pro hormone circulates normally in man,11-21 the present investigation was designed to determine if the N-terminus of this prohormone has any relationship to blood pressure, PRA, or plasma aldosterone concentrations in normotensive and hypertensive obese volunteers followed for 12 weeks on a weight reduction diet. ProANF 31-67, which circulates as a distinct entity after proteolytic cleavage from the midportion of the N-terminus,17 was also evaluated in the present investigation to determine its interrelationship, if any, to blood pressure, PRA, or plasma aldosterone in these volunteers. Materials and Methods Subjects. Eighteen obese subjects (5 men, 13 women),

25-74 years of age, were studied at the University of Arkansas for Medical Sciences Metabolic Management Clinic, Little Rock, AR. Criteria for inclusion in this study included obesity defined as a body mass index (kg/m2) greater than 25. All of these persons weighed more than 11 kg above their ideal body weight. Subjects were excluded if they had any disease other than hypertension or obesity. Specifically excluded were any persons with diabetes mellitus, cardiovascular disease other than hypertension, chronic lung disease, or psychiatric disease. Five of the female subjects had hypertension before weight reduction based on the criteria of at least three blood pressure measurements> 140 mm Hg systolic, >90 mm Hg diastolic, or a mean arterial pressure> 107 mm Hg, using a large thigh cuff. These elevated blood pressures were observed at three separate visits of at least 1 week apart before weight reduction was begun. To be included in the study, all subjects were required to have normal circulating concentrations of blood urea nitrogen, creatinine, glucose, electrolytes, uric acid, and serum thyroxine. In addition, a normal complete blood count, urinalysis, and electrocardiogram were required of all volunteers. All medications were stopped 2 weeks prior to entry in this study. Protocol. Based on their respective blood pressure measurements, patients were assigned to the hypertensive or normotensive (control) groups. All subjects were placed on a controlled diet (Optifast from Sandoz Nutrition, Minneapolis, MN) consisting of 420 cal/d given as 70 g protein, 30 g carbohydrate, and 2 g fat. In addition to 100% allowances for essential vitamins and minerals, the diet contained 922 mg/d of sodium and 1,955 mg/d of potassium. Patients were instructed THE AMERICAN JOURNAL OF THE MEDICAL SCIENCES

in low sodium fluid intake and dietary compliance. At the initial clinic visit after their history and physical examinations, the above laboratory tests were performed to exclude secondary forms of hypertension. The diet was administered on an outpatient basis in five divided portions per day. During each visit, body weight, arm circumference, and heart rate were measured. Blood pressure was determined three times at each visit by specially trained nurses and a physician, after 10 minutes of sitting rest using a large-sized (thigh) blood pressure cuff. At each visit, the physician measured the blood pressure, which was later repeated twice by either the physician or nurse. Averages of the three systolic and diastolic blood pressure measurements were recorded for each visit for both the hypertensive and nonhypertensive patients; the mean arterial pressure was calculated as the sum of the diastolic pressure and one third of the pulse pressure. Noncompliance with the diet was evaluated by serial measurement of body weight, urinary ketones, serum potassium, serum uric acid, and the dietitian/nurse/physician interview. Only persons who complied absolutely with the weight reduction diet were included in this study. Blood samples for electrolytes, plasma proANF 1-98 andproANF 31-67, PRA, and plasma aldosterone were obtained from the antecubital vein in the sitting position after 10-15 minutes of rest at each clinic visit. Samples for proANF 1-98, proANF 31-67, PRA, and aldosterone were collected in EDTA tubes, transported on ice, and immediately centrifuged at -4 cC. Radioimmunoassays (RIAs) to measure the N -terminus of the pro hormone were devised to aa 1-30 and 31-67 of the 126 aa prohormone as previously described by our laboratory. 17,18 G-50 sephadex gel permeation chromatography revealed that the proANF 1-30 RIA recognized a 10,000 m.w. peptide consistent with it measuring the whole N-terminus (ie, aa 1-98 of the prohormone), but lacking the C-terminus (ie, ANF).17 Thus, although the proANF 1-30 RIA was devised to synthetic amino acids 1-30 of the 126 aa pro hormone, it immunologically recognizes the whole 1-98 aa segment that comprises the N-terminus of the prohormone. The proANF 31-67 RIA, on the other hand, recognizes mainly (96%) a peptide of 3,900-4,000 m.w. from the midportion of the N-terminus of the prohormone consistent with aa 31-67 (actual proANF 31-67 m.w. = 3878).17 The proANF 31-67 RIA also recognizes a small amount (4%) of the whole N-terminus of the prohormone. The intraassay coefficient of variation for proANF 1-30 and proANF 31-67 RIAs were 4.8% and 5.3%, respectively. The interassay coefficient of variation was 8% for both proANFs 1-30 and 31-67 RIAs. Recovery of pure synthetic unlabeled proANF 1-30 and proANF 31-67 in these assays was 83.5 ± 13.2 (SD) and 100.9 ± 8.9 (SD) percent, respectively. The respective ICsos were 180, and 120 fmols/tube; the lowest detectable concentrations were 40 and 35 fmols for

3

ProANF 1-98 and Blood Pressure

proANF 1-30, and 31-67 RIAs, respectively. Serial dilution of pooled plasma has revealed excellent parallelism of standard and unknown in these assays.11,16-18 This investigation was approved (1/21/87) by the Human Use Committees of the University of Arkansas for Medical Sciences and the John L. McClellan Memorial Veterans Hospital. Plasma renin activity was determined by a RIA kit from Clinical Assays (Cambridge, MA), which uses a modification of the original method of Haber et a1. 22 Aldosterone was measured using the Biotecx Aldosterone Direct Radioimmunoassay Kit (Biotecx Labs, Friendswood, TX), which uses a modification of the original method of Mayes et al. 23 Statistical Analysis. All values expressed are the mean values ± SEM unless otherwise noted. Statistical analysis of the data between groups was performed by the two-way analysis of variance for repeated measurements. Correlation values of proANF 1-98, proANF 31-67, blood pressure, renin, and aldosterone were calculated by the Pearson correlation coefficient. Results

Body weight in the 18 subjects ranged from 40% to 250% (mean 75% ± 29%) above their calculated ideal body weight corrected for age and sex. The characteristics of the hypertensive and normotensive obese groups with respect to age, weight, body mass, and blood pressure are observed in Table 1. The normotensive and hypertensive groups were similar before beginning the weight loss program with only blood pressure in the two groups being significantly different (p < .005; Table 1). The amount of weight loss in each group was similar (ie, 18 ± 1.4 kg for the hypertensive group versus 16.5 ± 1.2 kg for the normotensive group). The serum sodiums and potassiums for individuals in both groups were the same at the beginning and throughout the study with the values at the beginning and at the end of 12 weeks of weight reduction observed in Table 1. Only subjects who complied with the weight reduction diet are included in the two groups. Body Weight. Weight loss in both the hypertensive and normotensive obese subjects was almost identical each week of the 12 weeks of the weight reduction diet. The amount of weight loss each week in each group has been illustrated previously in study 3 of the circulating concentration of ANF during a 12-week weight reduction diet. There was a significant weight loss in both groups by the end of the first week (p < 0.01). Comparisons between subsequent weeks of dieting revealed significant weight reduction from one period to the next (p < .01). After 12 weeks of the weight reduction diet, the hypertensive obese subjects had lost 18 ± 1.4 kg, whereas the normotensive obese patients had lost 16.5 ± 1.2 kg. At 8 weeks, both groups had lost exactly the same amount of weight (12 kg). Blood Pressure. Mean arterial blood pressure fell significantly after 1 week on weight reduction diet from

Table 1. Characteristics of the Normotensive and Hypertensive Obese Group Feature

Normotensive

Hypertensive

Number of subjects Before fasting Systolic blood pressure (mmHg) Diastolic blood pressure (mmHg) Mean arterial pressure (mm Hg) Weight (kg) Body mass index (g/m 2 )* Heart rate (beats/min) Age (yr) Serum sodium (mEq/L) Serum potassium (mEq/L) Serum creatinine (mEq/dL) 12-week values: Systolic blood pressure (mmHg) Diastolic blood pressure (mm Hg) Weight (kg) Heart rate (beats/min) Post-diet serum sodium (mEq/L) Post-diet serum potassium (mEq/L) Post-diet serum creatinine

13

5

126

±3

161

± 7

82

±2

92

± 3

96 102.3 37 80 43 142 4.1 0.9

±2 ± 6.1 ±2 ±2 ±4 ± 1 ± 0.1 ± 0.1

115 ± 106.8 ± 43 ± 82 ± 57 ± 141 ± 4.3 ± 1.1±

118

±3

143

76 ±3 86.3 ± 4.7 73 ±4 141

±1

4.0 ± 0.1 0.8 ± 0.5

2 20.6 8 6 7 1 0.1 0.2

± 3

81 ± 5 88.8 ± 19.2 72 ± 3 141

± 1

4.0± 0.6 0.8± 0.1

* Normal for body mass index is up to 25 kg/m2.

a baseline of 115 ± 2 mm Hg to 98 ± 6 mm Hg in the hypertensive group and from a baseline of 96 ± 2 mm Hg to 92 ± 2 mm Hg in the normotensive obese subjects (Figure 1). The reduction of systolic blood pressure was greater after 1 week of diet in the hypertensive group, with a systolic blood pressure decrease from 161 ± 7 mm Hg to 138 ± 8 mm Hg versus a mean systolic blood pressure decrease from 126 ± 3 mm Hg to 120 ± 3 mm Hg for the normotensive obese subjects. Likewise, the diastolic pressure decreased more in the hypertensive obese subjects after 1 week of weight reduction, with their mean diastolic blood pressure decreasing from 92 ± 3 mm Hg to 78 ± 6 mm Hg versus a decrease of 82 ± 2 mm Hg to 78 ± 2 mm Hg for the normotensive obese group. In both the normotensive and hypertensive obese groups, there was a rebound in mean arterial pressure on the second week of the weight reduction diet and then a slow decline in mean arterial pressure to the extent that at 12 weeks the mean arterial pressure .had decreased to that observed after 1 week of weight reduction (Figure 1). Systolic blood pressure after its dramatic decrease at week 1, increased in both groups at the second week to 143 ± 4 mm Hg and 124 ± 3 mm Hg, respectively, for the hypertensive and normotensive groups. At 12 weeks, systolic blood pressure was 143 ± 3 mm Hg in the hypertensive group, which was a decrease of over 18 mm Hg on the weight reduction

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4

January 1992 Volume 303 Number 1

McMurray and Vesely

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Plasma Aldosterone. Aldosterone concentration increased significantly in both the hypertensive and normotensive groups after week 1 of weight reduction (Figure 2). As one would expect, aldosterone and PRA correlated significantly, particularly over the last 10 weeks of weight reduction (hypertensive group: r = .54, p < 0.005; normotensive group: r = .75, p < 0.02). ProANF 1-98 had a significant inverse relationship to aldosterone over the 12 weeks of the study (r = .33; p < 0.01) in the normotensive group. Although the same inverse relationships for proANF 1-98 in the hypertensive group and ANF 31-67 in both groups appear to exist in Figure 2, it was not statistically demonstrable. Plasma Renin Activity. Plasma renin activity prior to study initiation was lower in the hypertensive obese group (1.7 ± 0.4 ng/ml/hr) than that ofthe normotensive obese group (2.6 ± 0.6 ng/mljhr), but this differ-

100

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Figure 2. Reciprocal change in plasma aldosterone (AId; mean ± standard error of the mean) versus plasma proANF 1-98 (N-terminus of ANF pro hormone; mean ± SEM) and proANF 31-67 (mean ± SEM) in hypertensive (HTN; upper portion of graph) and normotensive (NORM; lower half of graph) obese subjects with weight reduction. This relationship was not significant for proANF 31-67 and was significant (p < 0.01) only for the normotensive obese group but not the hypertensive group with respect to proANF 1-98 and plasma aldosterone when evaluated by Pearson's correlation coefficient.

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concentration as high as that found prior to initiation of weight reduction (Figure 1). Throughout the 12-week study period, the hypertensive group consistently had higher mean proANF 31-67 levels than the normotensive group, although the levels were not significantly different after week 1 (Figure 1). Like ANF3 and proANF 1-98 concentration, proANF 31-67 correlated strongly (r = .59; p < 0.001) with MAP throughout the 12-week study in both groups (Figure 1). Throughout the study, the circulating concentration of the N-terminus (proANF 1-98) correlated strongly with the C-terminus of the ANF pro hormone (ie, ANF; r = .83; p < .0001) and with the circulating concentration ofproANF 31-67 (r = .88; p < 0.0001) in both the hypertensive and normotensive groups. ProANF 3167 showed a significant inverse correlation to PRA in the hypertensive group (r = .66; p < 0.002), as well as the normotensive group (r = .29; p < 0.03; Figure 3). ProANF 31-67 levels did not significantly correlate with aldosterone in either group (Figure 2). ProANF 31-67 levels correlated strongly with the C-terminus (ANF) levels (r = .78; p < 0.0001); as well as with the circulating concentration of the N-terminus, proANF 1-98 (r = .83; p < 0.0001).

6

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Figure 3. Reciprocal change in plasma renin activity (PRA; mean ± standard error of the mean) versus the circulating concentrations ofproANF 1-98 (N-terminus of ANF prohormone) and proANF 3167 (mean ± SEM) during weight reduction in hypertensive (HTN) and normotensive (NORM) obese patients. Prior to initiation ofthe study, plasma renin activity was lower in the hypertensive group than in the normotensive group, but PRA increased significantly (p < 0.05) in both groups after 1 week of weight reduction when evaluated by the analysis of variance (ANOVA). Throughout the study, there was a significant inverse correlation of PRA to proANF 1-98 (r = .45, p < 0.001) and proANF 31-67 (r = .41, p < 0.001) when evaluated by Pearson's correlation coefficient. January 1992 Volume 303 Number 1

McMurray and Vesely

ence was not statistically significant. After 1 week of weight reduction, there was a significant (p < 0.05) increase in the PRA of both groups. Throughout the study, there was a significant inverse correlation of PRA to proANF 1-98 (r = .45; p < 0.001), and to proANF 31-67 (r = .41; p < 0.001). Plasma renin activity was inversely related to blood pressure during the first 2 weeks of weight reduction and then from 4 to 12 weeks, the diet followed the same downward trend as MAP (r = .48; p < 0.05). Discussion

The obese hypertensive subjects had significantly increased circulating concentrations of both the Nterminus of the ANF pro hormone (proANF 1-98) and proANF 31-67 from the midportion of the N-terminus compared to obese normotensive subjects prior to beginning weight reduction. Investigation of persons with essential hypertension have found similar elevated Nterminal ANF prohormone circulating concentrations. 19,20 The hypertension of persons with pheochromocytomas is associated with elevated N-terminal ANF prohormone levels, which decrease to normal with removal of the pheochromocytomas and normalization of blood pressure. 21 Since the N-terminus of the ANF prohormone contains the vasodilatory peptides Vessel dilator (proANF 31-67) and Long Acting Sodium Stimulator (proANF 1-30), one might have hypothesized that high blood pressure might be associated with an underproduction of the vasodilatory peptide containing N -terminus of the ANF prohormone. The present study demonstrating elevated N-terminal proANF circulating concentrations in obese hypertensives versus obese normotensive individuals and the previously reported elevated N-terminus levels associated with essential hypertension 19,20 and the hypertension of persons with pheochromocytomas21 indicates that there is no defect in production of the N-terminus of the ANF prohormone in any of these forms of hypertension. The C-terminus of the ANF prohormone consisting of aa 99-126 of the pro hormone (ie, ANF) is also elevated in the plasma of persons with pheochromocytomas,24 essential hypertension,25 and obese hypertensives versus normotensive obese individuals.3 Thus, there is no defect in production of any part of the ANF pro hormone in persons with hypertension, which could have theoretically been a cause of hypertension. The elevated circulating levels of the N -terminus and the C-terminus of the ANF prohormone in persons with hypertension suggests a defect in the target organ (ie, vasculature), with these vasodilatory peptides increasing to try to overcome the vasoconstriction that is present. The present investigation where blood pressure decreased with weight loss (without using any pharmacological drugs to lower blood pressure) and the circulating N-terminal atrial natriuretic prohormone simultaneously decreased and significantly correlated THE AMERICAN JOURNAL OF THE MEDICAL SCIENCES

with blood pressure over a 12-week period would further suggest: (1) that the defect leading to hypertension in these individuals is reversible, and (2) that the N -terminal vasodilatory peptides respond quickly and appropriately (ie, decrease when vasoconstriction is no longer present) to changes in blood pressure. The present data on proANF 31-67 would suggest that it also responds appropriately to changes in blood pressure. The initial reduction in proANF 1-98, proANF 3167, and blood pressure in the first week of this diet is most likely associated with the marked natriuresis observed previously in the first week of modified protein sparing diets by Sigler26 and by Marks et al. 27 The mechanism of this increased natriuresis is unknown but may be due, in part, to the following sequence of events. First, the total body sodium decreased due to the low salt intake (40 mmol) in the Optifast diet in combination with concomitant sodium restriction imposed by adherence to a ketogenic protein sparing diet. Whether the N -terminal ANF prohormone peptide Long acting sodium stimulator (proANF 1-30) or Vessel dilator (proANF 31-67) both with potent natriuretic properties9 or ANF is the cause of this natriuresis in the first week cannot be determined with certainty. However, a recent report suggests that ANF increases in the first 3 days of weight reduction 28 before decreasing at 1 week.3With respect to this point, it is important to note that the N-terminus and proANF 31-67 are released in response to sodium infusion to isolated cardiac atria in vitro29 and are released secondary to changes in intravascular volume in isolated atria30 and in vivo 18 simultaneously with ANF (ie, C-terminus of the prohormone). The combination of low salt intake with natriuresis should lead to a decreased plasma volume, which, in turn, should cause the atria to release less of the N-terminus and C-terminus of ANF prohormone. The decrease in release of the N-terminus would result in decreased circulating concentrations of proANF 1-98 and proANF 31-67 as found at 1 week in the present investigation. One interesting finding of the present investigation was that proANF 31-67 did not correlate in a reciprocal manner with the plasma aldosterone concentration as ANF previously did during weight reduction.3 This difference may be due to ANF's ability to inhibit angiotensin II stimulated aldosterone secretion while proANF 1-30 and proANF 31-67 do not inhibit angiotensin II -stimulated aldosterone secretion from isolated adrenal cells. 31 The N-terminal atrial natriuretic peptides and ANF appear to have different mechanisms of action with respect to sodium metabolism at the cellular level. Although proANF 1-30 andproANF 3167 are equally potent to ANF in causing sodium excretion and diuresis in vivo9 and proANF 31-67's inhibition of sodium transport in inner medullary collecting duct cells is half maximal at 10-11M, which is consistent with a physiological effect (and equal to ANF's effect),32 proANF 31-67, unlike ANF, may in-

7

ProANF 1-98 and Blood Pressure

hibit the Na+ /K+ -ATPase in the inner medullary collecting duct. 32 Thus, proANF 31-67 and ANF appear to have different mechanisms of action at the cellular level, although they both cause increased sodium excretion in vivo. 9 ProANF 31-67 and proANF 1-98 did correlate inversely with plasma renin activity in the present investigation. The effect ofproANF 31-67 and proANF 1-30 on PRA has not been determined in vitro. The present investigation would suggest that proANF 1-30 and proANF 31-67 in addition to their above effects may affect PRA because of the inverse ratio found in the N-terminal ANF peptides and plasma renin activity observed over the 12 weeks of the present investigation. References 1. DeBold AJ, Borenstein HB, Veress AT, Sonnenberg H: A rapid and potent natriuretic response to intravenous injection of the myocardial extract in rats. Life Sci 28:89-94, 1981. 2. Vesely DL: Atrial Natriuretic Hormones. Englewood Cliffs, NJ, Prentice Hall, 1991. 3. McMurray RW Jr., Vesely DL: Weight reduction decreases atrial natriuretic factor and blood pressure in obese patients. Metabolism 38:1231-1237, 1989. 4. Atarashi K, Mulrow PJ, Franco-Saenz R, Snajdar R, Rapp J: Inhibition of aldosterone production by an atrial extract. Science 224:992-994, 1984. 5. Chartier L, Schiffrin E, Thibault G: Effect of atrial natriuretic factor (ANF)-related peptides on aldosterone secretion by adrenal glomerulosa cells: Critical role of the intramolecular disulphide bond. Biochem Biophys Res Comm 122:171-174, 1984. 6. Kudo T, Baird A: Inhibition of aldosterone production in the adrenal glomerulosa by atrial natriuretic factor. Nature 312:756757,1984. 7. Burnett JC Jr., Granger JP, Opgenorth T J: Effects of synthetic atrial natriuretic factor on renal function and renin release. Am J PhysioI247:F863-F866, 1984. 8. Maack T, Marion DN, Camargo MJF, Kleinert HD, Laragh JH, Vaughan Ed Jr., Atlas SA: Effects of auriculin on blood pressure, renal function, and the renin-aldosterone system in dogs. Am J Med 77:1069-1075, 1984. 9. Martin DR, Pevahouse JB, Trigg DJ, Vesely DL, Buerkert JE: Three peptides from the ANF pro hormone NH 2 -terminus are natriuretic and/or kaliuretic. Am J Physiol 258:FI401-FI408, 1990. 10. Vesely DL, Norris JS, Walters JM, Jespersen RR, Baeyens DA: Atrial natriuretic prohormone peptides 1-30, 31-67, and 79-98 vasodilate the aorta. Biochem Biophys Res Commun 148:15401548,1987. 11. Winters CJ, Sallman AL, Vesely DL: Circadian rhythm of prohormone atrial natriuretic peptides 1-30, 31-67, and 99-126 in man. Chronobiol Int 5:403-409,1988. 12. Winters CJ, Sallman AL, Meadows J, Rico D, Vesely DL: Two new hormones: Pro hormone atrial natriuretic peptides 1-30 and 31-67 circulate in man. Biochem Biophys Res Commun 150:231236,1988. 13. !toh H, Nakao K, Mukoyama M, Sugawara A, Saito Y, Morii N, Yamanda T, Shiono S, Arai H, Imura H: Secretion of Nterminal fragment of a-human atrial natriuretic polypeptide. Hypertension II(Suppl 1):1-52-1-56, 1988. 14. Meleagros L, Gibbs JSR, Ghatei MA, Bloom SR: Increase in plasma concentrations of cardiodilatin (amino terminal pro-atrial natriuretic peptide) in cardiac failure and during recumbency. Br Heart J 60:39-44, 1988.

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15. Sundsfjord JA, Thibault G, Larochelle f', Cantin M: Identification and plasma concentrations of pro atrial natriuretic factor in man. J Clin Endocrinol Metab 66:609-610, 1988. 16. Vesely DL, Winters CJ, Sallman AL: Pro hormone atrial natriuretic pep tides 1-30 and 31-67 increase in hyperthyroidism and decrease in hypothyroidism. Am J Med Sci 297:209-215, 1989. 17. Winters CJ, Sallman AL, Baker BJ, Meadows J, Rico DM, Vesely DL: The N-terminus and a 4000 molecular weight peptide from the midportion ofthe N-terminus of the atrial natriuretic factor pro hormone each circulate in man and increase in congestive heart failure. Circulation 80:438-449, 1989. 18. Vesely DL, Norsk P, Winters CJ, Rico DM, Sallman AL, Epstein M: Increased release of the N-terminal and C-terminal portions of the prohormone of atrial natriuretic factor during immersioninduced central hypervolemia in normal humans. Proc Soc Exp Bioi Med 192:230-235, 1989. . 19. Buckley MG, Sagnella GA, Markandu ND, Singer DRJ, MacGregor GA: Immunoreactive N-terminal pro-atrial natriuretic peptide in human plasma: Plasma levels and comparisons with a-human atrial natriuretic peptide in normal subjects, patients with essential hypertension, cardiac transplant and chronic renal failure. Clin Sci 77:573-579, 1989. 20. Matsubara H, Mori Y, Takashima H, Inada M: Simultaneous measurement of a-human atrial natriuretic factor (hANF) and NH 2 -terminal fragment of proANF in essential hypertension. Am Heart J 118:494-499, 1989. 21. Vesely DL, Arnold WC, Winters CJ, Sallman AL, Rico DM: Increased circulating concentration of the N-terminus of the atrial natriuretic factor prohormone in persons with pheochromocytomas. J Clin Endocrinol Metab 71:1138-1146,1990. 22. Haber E, Koerner T, Page LB, Kliman B, Purnode A: Application of a radioimmunoassay for angiotensin I to the physiologic measurement of plasma renin activity in normal human subjects. J Clin Endocrinol Metab 29:1349-1355,1969. 23. Mayes D, Furuyama S, Kern DC, Nugent CA: A radioimmunoassay for plasma aldosterone. J Clin Endocrinol Metab 30: 682-685, 1970. 24. Vesely DL, Arnold WC, Winters CJ, Sallman AL, Rico DM: Increased circulating concentration of atrial natriuretic factor in persons with pheochromocytomas. Clin Exp Hypertens [A] 11:353-369, 1989. 25. Sugawara A, Nakao K, Sakamoto M, Morii N, Yamada T, Itoh H, Shiono S, Imura H: Plasma concentration of atrial natriuretic polypeptide in essential hypertension. Lancet 2:1426-1427, 1985. 26. Sigler MH: The mechanism of the natriuresis of fasting. J Clin Invest 55:377-387, 1975. 27. Marks P, Wilson B, DeLasalle A: Aldosterone studies in obese patients with hypertension. Am J Med Sci 289:224-228, 1985. 28. Maoz E, Sham iss A, Peleg E, Salzberg M, Rosenthal T: The role of atrial natriuretic peptide (ANP) in natriuresis of fasting [Abstract]. Intern Soc Hypertens, in press, 1990. 29. Dietz JR, Nazian SJ, Vesely DL: Atrial natriuretic factor (ANF) and proANF 1-98 during acute and chronic sodium loading in the rat. The Physiologist 33:A40, 1990. 30. Dietz JR, Nazian SJ, Vesely DL: Release of ANF proANF 1-98, and proANF 31-67 from isolated rat atria by atrial distension. Am J Physiol 260:Hl774-Hl778, 1991. 31. Denker PS, Vesely DL, Gomez-Sanchez CE: Effect of pro atrial natriuretic peptides 1-30, 31-67, and 99-126 on angiotensin II stimulated aldosterone production by calf adrenal cells. J Steroid Biochem 37:617-619, 1990. 32. Gunning M, Brenner BM, Otuechere G, Zeidel ML: ANP 3 1-67 inhibits transport-dependent oxygen consumption (Q02) in inner medullary collecting duct (IMCD) cells. J Am Soc Nephrol 1: 416,1990.

January 1992 Volume 303 Number 1