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Blunted Vascular and Renal Effects of Exogenous Atrial Natriuretic Peptide in Patients with Cushing’s Disease

Istituto Clinica Medica (C.S., A.M.) and Istituto Scienze Endocrine (B.A.), Università di Milano and Ospedale Maggiore Istituto Ricovero e Cura a Carattere Scientifico, 20122 Milan, Italy

Address all correspondence and requests for reprints to: Carla Sala, M.D., Centro Fisiologia Clinica e Ipertensione-Policlinico, Via F. Sforza 35, 20122 Milan, Italy. E-mail: carla.sala{at}unimi.it

	Abstract

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The role of atrial natriuretic peptide (ANP) in glucocorticoid hypertension is still controversial, as glucocorticoids enhance the secretion of ANP in vivo, but reduce its biological activity in vitro. In isolated cells, for example, the cGMP response to ANP is suppressed by dexamethasone. We tested the in vivo relevance of this observation by comparing the cGMP, endocrine, and renal responses to exogenous ANP in patients with Cushing’s disease (CD; n = 10) and in a matched group of essential hypertensives (EH; n = 8) and normotensive controls (N; n = 4). -human-ANP was infused at 0.01 µg/kg/min for 120 min with a 30-min recovery period; hormonal and arterial pressure measurements were performed at 30-min intervals, and renal parameters were measured at baseline and after infusion.

There was a 4-fold increase in plasma ANP in all groups, but the increments in plasma cGMP were about 50% lower in CD than in N and EH; urinary cGMP was similarly lower in CD (247 ± 61 vs. 529 ± 146 and 384 ± 54 nmol/150 min, respectively). This was associated with a reduced peak increase in hematocrit in CD (+2.6 ± 0.9%) compared with N (+6.6 ± 0.9%) and EH (+7.1 ± 0.7%; P < 0.05 CD vs. both); the diuretic and natriuretic effects of ANP were also lower in CD than in EH with very similar systemic pressure levels (382 ± 63 vs. 848 ± 130 mL/150 min, and 61 ± 14 vs. 113 ± 14 mmol/150 min, respectively; P < 0.05 for both).

The increments in plasma and urinary cGMP in response to physiological doses of ANP are thus blunted in CD patients compared with those in N and EH. This biochemical defect is associated with reduced capillary permeability and a lesser diuretic and natriuretic effect. These data are compatible with an impairment of the biological activity of ANP in glucocorticoid hypertension in humans.

	Introduction

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GLUCOCORTICOID HYPERTENSION is characterized by increased plasma volume and pressor responsiveness to vasoconstriction substances (1). High circulating levels of atrial natriuretic peptide (ANP) have been observed in experimental (2) and clinical (3, 4) conditions of glucocorticoid excess. This enhanced secretion of ANP is the result of the mechanical stimulus of atrial stretch (5) and also of the direct activation of ANP gene transcription by glucocorticoids in atrial and ventricular myocytes (6, 7). The compensatory increase in ANP might counteract the pressure/volume overload via a vasorelaxant, capillary-permeabilizing, diuretic and natriuretic effect (8). Experimental and clinical studies show that most of the vascular and renal actions of ANP are mediated by cGMP via activation of receptors coupled to guanylyl cyclase located on the membranes of endothelial, vascular smooth muscle, glomerular, and tubular cells (9). Despite this increase in ANP secretion in vivo, in vitro the biological activity of the peptide is reduced by glucocorticoids. A blunted response of the second messenger cGMP to ANP has been seen in vascular smooth muscle cells exposed to dexamethasone (10). This experimental finding has not been confirmed in vivo; Fujio et al. failed to observe any difference in the cGMP and renal responses to exogenous ANP in patients with Cushing’s syndrome compared with normotensive controls (11). In this study, however, ANP was infused at pharmacological doses that induced profound hemodynamic changes in both groups. Thus, despite the multiple interactions between the two systems observed in vivo and in vitro, the role of ANP in glucocorticoid hypertension is still not established. We, therefore, assessed the humoral, hemodynamic, and renal responses to physiological doses of ANP in patients with endogenous glucocorticoid excess.

	Subjects and Methods

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Patients

Ten patients with pituitary-dependent Cushing’s disease diagnosed on the basis of clinical features, radiological findings (magnetic resonance imaging and/or high resolution computed tomography scan of the pituitary), and standard hormonal criteria (high urinary free cortisol excretion, lack of ACTH/cortisol suppression after 1 or 2 mg/day dexamethasone and normal suppression after 8 mg/day, and positive ACTH and cortisol responses to CRH) were included into the study. Eight patients with essential hypertension (grade I–II) matched for age and body weight, were also studied. Four age-matched overweight subjects were included as controls. No patient was receiving pharmacological treatment, and all were on an ad libitum sodium diet; a 24-h urine collection was performed the day before the test. Informed consent was given by all participants to the study.

Protocol

Studies were performed in the morning after an overnight fast, with patients in the supine position for at least 30 min. After a priming dose of 100 ng, iv, -human-ANP (CLINALFA, Läufelfingen, Switzerland) diluted in 250 mL saline was infused at 0.01 µg/kg/min for 120 min, with subsequent 30-min recovery. Blood samples were collected at 30-min intervals from an antecubital vein of the contralateral arm kept open by slow saline infusion (2.0 mL/min). At the same time intervals, blood pressure was measured by sphygmomanometer, and pulse rate was recorded.

All subjects were asked to void before the study; at the end of the 150-min test, a second urine specimen was collected by spontaneous voiding.

Humoral determinations

Blood for ANP and cGMP levels was collected in prechilled tubes containing ethylenediamine tetraacetate (10 mmol/L) and centrifuged at 4 C, and plasma was stored at -40 C until assay. ANP was measured by RIA in plasma extracted on C₁₈ Sep-Pak cartridges (Waters Corp., Milford, MA) with a commercial antibody (Peninsula Laboratories, Inc., Merseyside, UK) and tracer (NEN Life Science Products, Boston, MA). cGMP was measured by RIA (RPA 525, Amersham Pharmacia Biotech, Aylsbury, UK) according to the method of Sagnella (12). PRA and aldosterone were measured by RIA (Technogenetics-Bouty, Milano, Italy). Plasma ACTH and serum cortisol were measured on unextracted samples by immunoradiometric assay (Nichols Institute Diagnostics, San Juan Capistrano, CA). The intra- and interassay variabilities of all assays were less than 10%. Packed cell volume was measured by microhematocrit; each sample was assessed twice. Plasma and urinary electrolytes were measured by a potentiometric method.

Statistical analysis

Results are expressed as the mean ± SEM. Comparison among groups was performed by ANOVA for repeated measures; post-hoc comparisons were performed by Bonferroni analysis. Pearson’s correlation was used for linear regression analysis. The area under the curve was calculated by the trapezoidal method. Statistical significance was set at P < 0.05.

	Results

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Baseline data

Table 1 shows baseline clinical, humoral, and renal data in patients with Cushing’s disease (CD), patients with essential hypertension (EH), and normotensive overweight subjects (N). In CD patients no gender-related differences were observed either at baseline or after ANP.

Hematocrit in CD was lower than that in the other two groups and was directly related to cortisol (r = 0.64; P < 0.05) and inversely to ANP (r = -0.59; P < 0.05).

Plasma ANP and cGMP were not significantly different among the three groups. A correlation between plasma ANP and cGMP was present in EH and N (r = 0.90 and 0.94, respectively; P < 0.01), but not in CD (r = -0.29; P = NS)

Plasma sodium (143 ± 1, 143 ± 1, and 142 ± 2 mmol/L) as well as potassium (4.0 ± 0.1, 3.8 ± 0.1, and 3.9 ± 0.1 mmol/L) were very similar in CD, EH, and N, respectively.

Diuresis, natriuresis, and kaliuresis were not different in the three groups, whereas cGMP excretion was lower in CD than in N.

ANP infusion

There was a 4-fold increase in plasma ANP in all groups (Fig. 1A), but plasma cGMP increments were significantly lower in CD at all times of the study (Fig 1B). Table 2 shows the areas under the curve for plasma ANP and cGMP.

View larger version (21K): [in this window] [in a new window]   Figure 1. Plasma ANP (A) and cGMP (B) at baseline and during ANP infusion N, EH, and CD. Values are the mean ± SEM. *, P < 0.01, CD vs. both N and EH. For similar circulating levels of ANP throughout the study, the increments in plasma cGMP were significantly lower in CD than in EH and N.

Systolic pressure tended to decrease, although not significantly, in EH and N (to 141.9 ± 3.6 and 130.6 ± 7 mm Hg, respectively), whereas it was unchanged in CD (146.3 ± 6.5 mm Hg). Diastolic pressure and heart rate were unchanged throughout the study in all groups.

PRA decreased similarly in CD and EH (to 0.4 ± 0.1 and 0.5 ± 0.1 µg/L/h, respectively; P < 0.05 vs. baseline for both), but not for N (0.8 ± 0.3 µg/L/h). Aldosterone decreased to a similar extent in all groups (to 187 ± 37 pmol/L in CD, 133 ± 15 in EH, and 124 ± 4 in N; P < 0.05 vs. baseline for all). Cortisol was unchanged in CD (679 ± 78 nmol/L), whereas it increased in EH and N (to 474 ± 56 and 533 ± 225 nmol/L, respectively; P < 0.05 for both).

Plasma sodium was unchanged, whereas potassium decreased similarly in all groups, to 3.7 ± 0.1 in CD, 3.7 ± 0.1 in EH, and 3.6 ± 0.1 mmol/L in N (P < 0.05 vs. baseline for all).

Urinary cGMP excretion was lowest in CD; the values reached statistical significance in comparison with N (P < 0.01), but not with EH (P = 0.07; Fig. 3C). Both diuresis and natriuresis were lower in CD than in EH despite very similar systemic arterial pressure (Fig. 3, A and B). Potassium excretion was not different among the three groups (12 ± 2 in CD, 29 ± 13 in EH, and 15 ± 5 mmol/150 min in N).

View larger version (18K): [in this window] [in a new window]   Figure 3. Urine volume (A), urinary excretion of sodium (B), and cGMP (C) at the end of ANP infusion in N, EH, and CD. Values are the mean ± SEM. *, P < 0.05.

	Discussion

Top Abstract Introduction Subjects and Methods Results Discussion References

ANP may increase capillary permeability (14) by interacting with specific binding sites on endothelial cells, where most circulating cGMP is generated (15). A hemoconcentrating effect of ANP and a decrease in plasma volume have also been demonstrated in nephrectomized animals (16). The reduced increments in hematocrit observed in patients with Cushing’s disease are compatible with an impaired capacity of ANP to shift fluid from the intravascular to the interstitial space; this difference can hardly be attributed to the renal loss of fluids, as diuresis, at least in the normotensive subjects, was similar. It is also unlikely that hypertension per se is responsible for the blunted effect of ANP on endothelial cells in patients with Cushing’s disease, because in essential hypertensives with indistinguishable arterial pressure, a more pronounced increase in plasma cGMP and hematocrit occurred.

A large part of the renal effects of ANP is mediated by receptors coupled to guanylyl cyclase and localized in the glomeruli and tubular cells, where most of the urinary cGMP is generated (17). The diuretic and natriuretic effects of ANP are related to systemic pressure and have been shown to be greater in essential hypertensives than in normotensives (18). In our study both diuresis and natriuresis are markedly reduced in patients with Cushing’s disease compared with those in essential hypertensives despite similar arterial pressure values.

Our data are compatible with a decreased production of cGMP in endothelial and renal cells, where this nucleotide is synthesized via the activation of type A receptors coupled to guanylyl cyclase (19). Three types of natriuretic peptides receptors have been identified: the biologically active A and B receptors coupled to guanylyl cyclase and the clearance C receptors (20).

Glucocorticoids have been shown to control gene expression of these receptors on endothelial (13), vascular smooth muscle (10), and glomerular (21, 22) cells. Thus, glucocorticoids not only activate gene transcription of ANP in cardiac myocytes, but also regulate the number and type of ANP receptors expressed on target cells.

The blunted response of cGMP to ANP may result from a decreased number of type A receptors on vascular and renal cells in patients with Cushing’s disease; this hypothesis, however, is not supported by the experimental evidence that dexamethasone instead stimulates gene transcription of these receptors in vascular smooth muscle (23) and renal (22) cells. In addition, down-regulation of A receptors, reflecting a chronic increment in agonist levels, can be excluded, as baseline values of ANP in patients with Cushing’s disease were not different from those in other groups. Similarly, metabolism of cGMP via steroid-induced activation of phosphodiesterases is not supported by the in vitro evidence that the response of cGMP to ANP remains suppressed in the presence of a phosphodiesterase inhibitor (10). A more likely explanation for our data are reduced activation of the biologically active A receptors reflecting enhanced clearance of ANP by type C receptors; this is supported by the in vitro observation that gene transcription of C receptors is up-regulated by dexamethasone in endothelial (13) and mesangial (21) cells. Type C receptors represent more than 95% of the total ANP receptor population and account for more than 55% of ANP clearance by an endocytotic process (19). Clearance receptors are widely distributed on ANP target cells, including renal, endothelial, and smooth muscle cells; more recently C receptors have also been demonstrated to be highly expressed in adipocytes (24). The role of adipose tissue in promoting ANP clearance in conditions of glucocorticoid excess need further investigation; in this regard, it should be pointed out that in our study major differences in fat distribution can be excluded, as the three groups were thoroughly matched for body mass index. It is worth noting that adipose tissue is not only a clearance, but also a target tissue for ANP, as in human adipocytes ANP has been recently shown to exert a cGMP-mediated lypolytic effect (25).

The enhancement of ANP clearance in patients with Cushing’s disease is not inconsistent with our finding that plasma levels of ANP were similar in the three study groups. In experimental and clinical studies, the biological activity of ANP is increased by the inhibitors of neutral endopeptidase, the enzyme that represents the alternative clearance pathway of ANP, in the absence of changes in circulating levels of ANP (26).

The physiological doses of ANP used in the present study induced only minor changes in arterial pressure and heart rate, as previously described in normotensives (27) and essential hypertensives (18). In contrast, the pharmacological doses of ANP used by Fujio et al. (11) induced a profound decrease in arterial pressure and a reactive tachycardia in patients with Cushing’s syndrome and in normotensive controls.

In conclusion, a blunting of the cGMP-mediated, capillary-permeabilizing, diuretic and natriuretic effects of ANP has been observed at physiological doses of ANP in patients with Cushing’s disease. If the role of ANP is to prevent volume overload by shifting fluids from intravascular to interstitial space and by increasing renal losses, this role appears to be impaired by glucocorticoids. In the long term, defective control of intravascular volume may play a role in the pathogenesis of glucocorticoid hypertension.

View larger version (19K): [in this window] [in a new window]   Figure 2. Percent increments in hematocrit during ANP infusion in N, EH, and CD. Values are the mean ± SEM. *, P < 0.05, CD vs. both N and EH.

Revised January 18, 2001.

Accepted January 26, 2001.

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