J. Endocrinol. Invest. 13. 133-137, 1990

Atrial natriuretic peptide in Cushing's disease G. Opocher, S. Rocco, G. Carpene, F. Pedini, M. Scarante, R. Milani, M. Boscaro, and F. Mantero* Istituto di Semeiotica Medica, Universita di Padova, 35100 Padova, and *Istituto di Clinica Medica III, Universita di Catania, 95100 Catania, Italy , ABSTRACT. Atrial Natriuretic Peptide (ANF), is secreted by atrial myocytes in response to atrial stretch. Its plasma levels have been found elevated in conditions leading to salt and fluid repletion and consequent atrial distention. Recently, it has been demonstrated that dexamethasone can enhance ANF secretion, by acting on ANF gene expression and mRNA synthesis. High plasma levels of ANF have been observed in normal man after administration of cortisol and ACTH. In the case of glucocorticoid excess, as in Cushing's disease, limited and conflicting data are available. Therefore, we measured ANF basal values and ANF response to postural changes and volume

expansion in eight patients with Cushing's disease. In our patients ANF values were higher than normals. ANF responded to volume expansion, 47.8 ± 5.1 pg/ml before sodium load and 69.9 ± 7.0 pg/ml after sodium load, and changed minimally after postural manoeuvres, 47.3 ± 3.2 pg/ml supine and 41.7 ± 5.1 pg/ml upright. These data indicate that ANF secretion is enhanced in Cushing's disease, and its regulation is partially altered. Since in this condition hypervolemia has not been certainly demonstrated, a direct relationship between elevated ANF and glucocorticoid excess could be suggested.

INTRODUCTION Atrial natriuretic peptide (ANF) is a recently discovered peptide hormone produced by atrial myocytes, involved in sodium and plasma volume regulation. Its secretion is mainly stimulated by factors leading to atrial distention, such as volume expansion, supine posture, head-out water immersion, and high sodium diets. In diseases characterized by chronic volume expansion (heart and renal failure, primary aldosteronism) ANF levels are elevated (1). The secretion of ANF appears to be stimulated by glucocorticoids. In fact, administration of pharmacological doses of dexamethasone can increase ANF and ANF mRNA levels in rat atrial cells (2). This effect could be due to a direct effect on gene expression since it is also present in vitro in cultured atrial myocytes of rats (3). Moreover, the recently demonstrated ventricular ANF mRNA is also enhanced by dexamethasone (4). Other Authors, how-

ever, do not confirm a stimulating effect of dexamethasone (5, 6), but still suggest a glucocorticoid responsive ANF gene (6). In normal subjects, cortisol or ACTH long term administration can enhance ANF plasma levels (7), but in condition of chronic glucocorticoid hypersecretion, such as in Cushing's syndrome, contradictory results have been obtained (8-10). Therefore, we measured ANF plasma levels in basal conditions, after postural changes, and after saline load, in 8 patients affected by Cushing's disease due to a pituitary ACTH-secreting tumor. MATERIALS AND METHODS We studied 8 patients affected by pituitary-dependent Cushing's disease (age 36 ± 3.8 yr), The diagnosis was supported by the effect of oCRH injection and dexamethasone suppression test and pituitary or adrenal CAT imaging, Any therapy was withdrawn for at least 2 months prior to the study, The clinical pattern of the patients is described in Table 1. The patients fasted overnight and remained in a recumbent position for at least 2 h before the study. The study was performed on two diHerent days. On the first day, blood samples for plasma renin activity

Key-words: ANF, Cushing's disease, cortisol, adrenal cortex. Correspondence: Dr. G. Opocher, Istituto di Semeiotica Medica, via Ospedale 105, 35100 Padova, Italy.

Received March 7, 1989; accepted November 8, 1989.

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G. Opocher, S. Rocco, G. Carpene, et al.

evaporator. The recovery of 125 1 alpha human ANF was 79.1 ± 13.9, n 48. The radioimmunoassay was performed using 125 1 alpha human ANF from Amersham and a specific anti alpha human ANF from Peninsula Lab. The sensitivity (ED 80) was 9.0 ± 1.2 pg, the interassay C.v. was 8.8 ± 4.6%, n = 38, and the interassay C.v. was 14.4 ± 3.0%, n = 38. Normal values: supine 25 ± 12, upright 16 ± 7 pg/ml n = 16, age 33 ± 2.4. Plasma and free urinary cortisol were measured by RIA (OPC, U.S.A.). DOC, 18-0H-DOC, corticosterone and 18-0H-corticosterone were measured by RIA after simultaneous HPLC separation (11 ). Statistical analysis: the results are expressed as Mean ± SE. One way analysis of variance was performed using STATVIEW software.

(PRA), aldosterone and ANF were drawn after 2 h of supine posture (supine) and after 2 h of upright posture (upright). ANF was measured again 15 min after return to the supine position. On the second day the patients, in the recumbent position, received an infusion of 0.9% saline at a rate of 8.4 ml/min for 4 h. PRA, aldosterone and ANF were measured before and after saline load. Plasma aldosterone (normal values: supine 4-12, upright 10-30 ng/ dl) and PRA (normal values: supine 1-3, upright 2-6 ng/ml/3h) were measured by radioimmunoassay ALOOCTK2 and RENCKT, respectively, Sorin Biomedica, Italy). Plasma ANF was measured by radioimmunoassay after Sep Pak C18 cartridges purification. The following procedure was used: venous blood was collected in iced polypropylene tubes containing Na-Heparin and stored at -20 C after centrifugation at 4 C. Plasma samples were applie.d to cartridges previously activated by 2 ml of Methanol/Trifluoroacetic acid 0.1 %, followed by 2 ml of Water /Trifluoroacetic acid 0.1 % and 2 ml of Phosphate Buffer (containing aprotinin 500 U.l.CH/ml). Cartridges were washed with 4 ml of Buffer, 4 ml of Water /Trifluoroacetic acid 0.1 %, 2 ml of Water/Trifluoroacetic acid 0.1% and Methanol/Trifluoroacetic acid 0.1 % 65: 35 and then eluted with 2 ml of Methanol/Trifluoroacetic acid 0.1 %. The extract was dried overnight with a Speed Vac

RESULTS PRA values were within the normal range both in the supine (1.4 ± 0.3 ng/ml/3h) and in the upright position (4.7 ± 0.6 ng/ml/3h). Saline load significantly suppressed PRA values from 1.3 ± 0.2 to 0.6 ± 0.1 ng/ml/3h, p < 0.05. Plasma aldosterone values were at the lower limit of the normal range (4.4 ± 0.7 ng/dl) and were significantly responsive to upright posture (12.0 ± 2.3 ng/dl, p < 0.05). Plasma aldosterone

Table 1 - Clinical data of the patients with Cushing's disease. No.

1 2 3 4 5 6 7

8

1 2 3

4 5 6 7

8

BP

K+

(mm/Hg)

(mmol/L) (3.5-5)

PRA (ng/ml/3h) (1-2)

ur. cortisol (J.Lg/24h) (60-120)

ANF [pg/ml (upright)) (9-23)

M

145/90 170/110 150/110 160/90 180/110 150/110 150/120 140/80

3.9 3.9 4.1 4.1 3.6 4.4 4.1 3.8

0.62 1.56 3.41 1.18 1.72 1.33 0.65 0.70

528 225 291 406 744 131 189 906

58 68 39 37 37 25 42 27

DOC (ng/dl) (5-30)

18-0H-DOC (ng/dl) (0-10)

Corticosterone (ng/dl) (100-3000)

18-0H-corticosterone (ng/dl) (10-35)

pI. cortisol (J.Lg/dl) (5-20)

aldosterone [ng/dl (supine)) (4-12)

4.8 17.4 45.3 21.5 15.0 49.0 7.1 9.9

25.3 4.5 13.7 9.8 8.9 28.5 6.2 2.7

404 670 1615 1031 1799 1040 1582 359

10.7 20.6 25.9 35.3 29.5 270 34.6 10.6

30.5 13.1 12.6 21.3 26.4 15.8 29.3 27.9

3.0 6.1 7.5 6.4 3.5 3.4 4.0 1.3

Age (yr) normal values

Sex

20 51 36 32 39 38 49 23

M

F

F

F F F

F

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ANP in Cushing's disease

was suppressed by saline load (from 4.5 ± 0.7 to 2.8 ± 0.3, P < 0.05). ANF values (Fig. 1) were elevated with little difference between the supine position, the upright position and after 15 min of supine posture, 47.3 ± 3.2, 41.7 ± 5.1 and 50.3 ± 4.5 pg/ml, respectively (p< 0.0003, supine, p< 0.002, upright, vs control subjects). Saline load significantly stimulated ANF secretion from 47.8 ± 5.1 to 69.9 ± 7.0 pg/ml, p< 0.05.

are significantly higher than in normal subjects and are similar to the levels we found in patients with primary aldosteronism (11 ). Limited and contradictory results of ANF levels in Cushing's syndrome due to adrenal adenoma have been reported. ANF levels were elevated in one case but normal in three others (8), while other Authors (9) have shown high ANF levels in four patients with this syndrome. Yamaji et al. (10) recently demonstrated in patients with Cushing's syndrome (12 Cushing's disease and 6 adrenocortical adenoma) increased plasma levels of ANF, which were correlated positively with plasma cortisol. The high levels of ANF in Cushing's disease could be due to the hypersecretion of adrenal steroids with sodium retaining action. In fact, the increase of ANF observed after ACTH and cortisol administration in normal man (7) has been explained by the plasma volume-dependent atrial distention. Recently, however, it has been observed that chronic administration of prednisone (13) is associated with a significant elevation of ANF levels without significant sodium and fluid retention. Despite the possibility of a glucocorticoid-induced reduction of the metabolic clearance of ANF, the higher plasma levels of ANF can be accounted to atrial or ventricular hypersecretion. Volume expansion is not usually present in Cushing's disease (14). In our patients, mineralocorticoid hormones (aldosterone, DOC, B, 18-0HDOC and 18-0H-B) were within the normal range in most cases (Table 1) and plasma renin activity was never suppressed.' Although it is well known that glucocorticoids can increase PRA, its significant suppression after acute saline load argues against a significant volume expansion. Furthermore, a sustained mineralocorticoid activity does not seem to be present in our patients. ANF has been reported to be high or slightly elevated in some forms of hypertension (9) as a consequence of increased central volume and atrial distention. However, hypertension was present only in 5 patients (Table 1 ) while in 3 patients with normal blood pressure ANF was elevated. Hypertension and ANF were not found to be always associated in our patients. We did not observe a clear-cut postural response of ANF as in normal subjects (1 ). This can be related to a decrease in storage of ANF during chronic stimulation or, in alternative, to a partial insensitivity to volume manipulation in Cushing's disease: ANF increased significantly after a greater degree of volume expansion, such as after

DISCUSSION In this study we demonstrated that in humans, chronic glucocorticoid hypersecretion due to Cushing's disease is associated with elevated ANF values both in the supine and in the upright position. They

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Atrial natriuretic peptide in Cushing's disease.

Atrial Natriuretic Peptide (ANF), is secreted by atrial myocytes in response to atrial stretch. Its plasma levels have been found elevated in conditio...
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