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The Journal of Clinical Endocrinology & Metabolism Vol. 83, No. 1 253-257
Copyright © 1998 by The Endocrine Society

Original Studies

Proadrenomedullin N-Terminal 20 Peptide Inhibits Aldosterone Secretion of Human Adrenocortical and Conn’s Adenoma Cells: Comparison with Adrenomedullin Effect

P. G. Andreis, C. Tortorella, G. Mazzocchi and G. G. Nussdorfer

Department of Anatomy, University of Padua, I-35121 Padua, Italy

Address correspondence and requests for reprints to: Professor G. G. Nussdorfer, Department of Anatomy, Via Gabelli 65, I-35121 Padua, Italy. E-mail: ggnanat{at}ipdunidx.unipd.it


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
Adrenomedullin (ADM) and proadrenomedullin N-terminal 20 peptide (PAMP) are two vasoactive peptides, which are highly expressed in human adrenal gland. Autoradiography showed the presence of abundant [125I]ADM and [125I]PAMP binding sites in both the outer cortex and medulla of human adrenals. ADM, but not PAMP binding was completely displaced by the specific CGRP1 receptor antagonist CGRP(8-37). ADM and PAMP concentration-dependently inhibited angiotensin-II (ANG-II)-stimulated, but not basal aldosterone secretion of dispersed human adrenocortical cells. PAMP was significantly more potent than ADM (IC50, 0.98 x 10-11 vs. 3.16 x 10-9 mol/L). CGRP(8-37) abolished the inhibitory action of ADM, without affecting that of PAMP. Qualitatively analogous findings were obtained using aldosteronoma dispersed cells. However, tumor cells were more sensitive than normal adrenocortical cells (IC50 were 1.32 x 10-12 and 1.51 x 10-9 mol/L for PAMP and ADM, respectively). Moreover, PAMP was found to also depress basal aldosterone secretion (IC50, 4.27 x 10-11 mol/L). Neither basal nor ANG-II-stimulated cortisol production by both normal and tumorous adrenocortical cells was altered by ADM or PAMP. Collectively, these findings confirm that ADM (CGRP1) and PAMP receptors are present in the human outer adrenal cortex and allow us to draw the following conclusions: 1) because of its potency, PAMP may a better candidate for being considered a physiological regulator of aldosterone secretion than ADM; and 2) under pathological conditions, both peptides may be capable of reversing overproduction of aldosterone.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
ADRENOMEDULLIN (ADM) is a hypotensive peptide synthesized and secreted by adrenal medulla of several mammalian species. Human ADM is a 52-amino acid peptide, possessing a 6-membered ring structure formed by the disulfide bridge between adjacent cysteine residues, which is produced as a part of a 185-amino acid prohormone, named preproadrenomedullin (95-146 fragment). Preproadrenomedullin also contains a unique 20-amino acid sequence in its N-terminus (after the cleavage of the signal peptide), exerting only a transient hypotensive action and called proadrenomedullin N-terminal 20 peptide (PAMP) (for review, see Refs. 1, 2).

Like other regulatory peptides contained in adrenal medulla (for review, see Ref.3), ADM was found to affect the secretory activity of the adrenal cortex; it specifically inhibits angiotensin-II (ANG-II)- and potassium-stimulated aldosterone production by dispersed rat zona glomerulosa cells, probably by impairing Ca2+ influx (4, 5). We have recently confirmed this finding in humans, but the minimal effective concentration of ADM was relatively elevated (10-7 M) (6). In both rats and humans, ADM appears to exerts its aldosterone antisecretagogue effect, acting through the subtype 1 of CGRP receptors (5, 6).

Abundant [125I]PAMP binding sites have been demonstrated in the rat adrenals (7), and evidence has been provided that PAMP, like ADM, inhibits aldosterone stimulating effect of ANG-II (8). Therefore, it seemed worthwhile to study the distribution of PAMP binding sites in the human adrenal and the effect of the peptide on the aldosterone secretion of human normal and tumorous adrenocortical cells.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Reagents

Human ADM(1-52), human PAMP, and the CGRP1 receptor antagonist CGRP(8-37) (5) were purchased from Peninsula Labs (Merseyside, UK). [125I]ADM(1-52) and [125I]PAMP (specific activity, 2000 Ci/mmol) were obtained from Amersham Labs (Amersham, UK), medium 199 from DIFCO (Detroit, MI), and ANG-II, human serum albumin (HSA) and other laboratory reagents from Sigma Chemical Co. (St. Louis, MO). RIA kits for aldosterone and cortisol were purchased by IRE-Sorin (Vercelli, Italy).

Adrenal glands and tumors

Fragments of adrenal glands were obtained from six consenting adult patients (40–50 yr old) undergoing unilateral nephrectomy for kidney cancer. Starting from two weeks before surgery, patients were kept on a normal diet. Only patients not requiring medications able to alter adrenal function were recruited. Portions of the head and tail of each adrenal, which, respectively, contain and do not contain medullary chromaffin tissue (6) were removed. Fragments of Conn’s adenomas were obtained from two other patients displaying high basal levels of circulating aldosterone. The study protocol followed the local Ethical Committee guidelines for human studies.

Autoradiographic studies

Adrenal head fragments were immediately frozen at -30 C by immersion in isopentane and stored at -80 C. Frozen 10–15 µm thick sections were cut in a Leitz 1720 Digital cryostat (Leitz, Wetzlar, Germany) at -20 C, and autoradiographic procedures were those previously described (9). ADM and PAMP binding sites were labeled in vitro by incubation for 120 min with 10-7 mol/L [125I]ADM(1-52) or 10-9 mol/L [125I]PAMP; nonspecific binding was determined by addition of an excess of cold ADM or PAMP. The selectivity of [125I]ADM(1-52) and [125I]PAMP binding was checked by addition of 10-7 mol/L PAMP and ADM(1-52), respectively, or 10-5 mol/L CGRP(8-37).

Steroid secretion studies

Adrenal tail and adenoma fragments were employed to obtain dispersed cell preparations by collagenase digestion and mechanical disaggregation, as detailed previously (6). Dispersed cells obtained from each adrenal and tumor were placed in medium 199 and Krebs-Ringer bicarbonate buffer with 0.2% glucose, containing 5 mg/mL HSA, and incubated (3 x 104 cells/mL, in replicates of three each) as follows: 1) ADM(1-52) or PAMP (from 10-14 to 10-6 mol/L) alone or in the presence of 10-9 mol/L ANG-II; and 2) 10-5 mol/L CGRP(8-37) plus 10-7 mol/L ADM(1-52) or 10-9 mol/L PAMP. The incubation was carried out for 90 min in a shaking bath at 37 C in an atmosphere of 95% O2—5% CO2. Aldosterone and cortisol were extracted from the incubation media and purified by high-pressure liquid chromatography (6). Their concentrations, measured by commercial RIA kits, were: ALDO-CTK2: sensitivity, 5 pg/mL; intra- and interassay variations, 7.5% and 8.8%, respectively; cortisol-RIA: sensitivity, 30 pg/mL; intra- and interassay variations, 6.0% and 7.2%, respectively.

Statistics

Data obtained from each adrenal gland or Conn’s adenoma were averaged and expressed as mean ± SEM of three or two separate experiments (three adrenals from three patients or two tumors from two patients). The statistical comparison of results was performed using ANOVA, followed by the multiple range test of Duncan.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Autoradiography

Autoradiography gave evidence of [125I]ADM and [125I]PAMP binding sites in both the outer portion of the cortex (including subcapsular zona glomerulosa) and adrenal medulla (Fig. 1Go, A and C), which were completely displaced by the addition of an excess of unlabeled peptides (data not shown). Cold ADM and PAMP did not alter [125I]PAMP and [125I]ADM binding, respectively (data not shown). Conversely, CGRP(8-37) completely eliminated [125I]ADM binding, without apparently affecting that of [125I]PAMP (Fig. 1Go, B and D).



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  		Figure 1. Autoradiographs of ematoxylin-eosin-stained frozen sections of human adrenal cortex incubated with [125I]ADM (A) or [125I]PAMP (C). Binding sites are more abundant in the outer adrenal cortex (AC) and adrenal medulla (AM). CGRP(8-37) completely displaced the binding of [125I]ADM (B), but not [125I]PAMP (D). The arrow heads indicate the gland capsule, and the arrow points the cortico-medullary border. V, Medullary veins; *, extracapsular vessel. x140.

 
Steroid secretion

Neither ADM nor PAMP affected basal aldosterone secretion of dispersed adrenocortical cells (Fig. 2Go). Conversely, both peptides inhibited ANG-II-stimulated aldosterone secretion in a concentration-dependent manner (Fig. 3Go). PAMP was more effective than ADM: minimal effective concentration, 10-12 vs. 10-8 mol/L; maximum inhibition, 48% vs. 22%; IC50, 0.98 ± 0.32 (SD) x 10-11 vs. 3.16 ± 0.87 x 10-9 mol/L (P < 0.01). CGRP(8-37) abolished the inhibitory effect of 10-7 mol/L ADM on ANG-II-enhanced aldosterone secretion, without per se evoking any sizable response. Conversely, CGRP(8-37) did not alter the effect of PAMP (Fig. 4Go).



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  		Figure 2. Effects of ADM and PAMP on basal aldosterone production by dispersed human adrenocortical (upper panel) and Conn’s adenoma cells (lower panel). Data are means ± SEM of three or two separate experiments. +, P < 0.05 vs. the respective control value (C).

 


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  		Figure 3. Effects of ADM and PAMP on 10-9 mol/L ANG-II-stimulated aldosterone production by dispersed human adrenocortical (upper panel) and Conn’s adenoma cells (lower panel). Data are means ± SEM of three or two separate experiments. +, P < 0.05 and *, P < 0.01 vs. the respective control value (C).

 


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  		Figure 4. Effect of CGRP(8-37) (10-5 mol/L) on ADM and PAMP inhibitory action on ANG-II (10-9 mol/L)-stimulated aldosterone production by dispersed human adrenocortical cells. Data are means ± SEM of three separate experiments. *, P < 0.01 vs. the respective basal value (B); A, P < 0.01 vs. the respective control value.

 
Baseline aldosterone secretion of Conn’s adenoma cells was higher than that of normal adrenocortical cells. PAMP exerted a significant inhibitory action on basal aldosterone production by tumorous cells (minimal effective concentration, 10-11 mol/L; maximum inhibition, 45%; IC50, 4.27 ± 1.83 [SD] x 10-11 mol/L) (Fig. 2Go). PAMP suppressed aldosterone response to ANG-II more efficiently than ADM (Fig. 3Go): minimal effective concentration, 10-12 vs. 10-8 mol/L; maximum inhibition, 45% vs. 20%; IC50, 1.32 ± 0.41 (SD) x 10-12 vs. 1.51 ± 0.45 x 10-9 mol/L (P < 0.01). The comparison of IC50 concentrations showed that both ADM and PAMP were more effective in aldosteronoma than in normal adrenocortical cells (P < 0.05).

Neither basal nor ANG-II stimulated cortisol secretion of both types of cells was significantly changed by ADM and PAMP (data not shown).


   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
Our present findings confirm that rather elevated concentrations of ADM are able to partially inhibit aldosterone, but not cortisol response of human adrenocortical cells to ANG-II, acting through the CGRP1 receptors (6). They also provide the first autoradiographic demonstration that CGRP(8-37)-displaceable ADM binding sites are present almost exclusively in the outer cortex and adrenal medulla.

In keeping with the results of earlier studies carried out in the rat (9), we show that PAMP is much more potent than ADM in suppressing aldosterone response to ANG-II of human adrenocortical cells. The autoradiographic distribution of PAMP binding sites is superposable to that of ADM, but PAMP binding is not displaced by either ADM or CGRP1-receptor ligand. This observation, coupled with the ineffectiveness of CGRP(8-37) to counteract the antimineralocorticoid action of PAMP, strongly suggests that this peptide acts via specific receptors.

Our present investigation does not clarify whether PAMP, like ADM (6), exerts its direct inhibitory action on human adrenocortical cells by impairing ANG-II-stimulated Ca2+ influx. In this connection, it must be recalled that there is now a general consensus that an intra-adrenal renin-angiotensin system, mainly located in the capsule-zona glomerulosa, plays an important role in the paracrine control of aldosterone secretion (10). Hence, the possibility that the proadrenomedullin-derived peptides may interfere with this local system awaits exploration.

Both ADM and PAMP are present in the blood, but their concentrations, at least under physiological conditions, are in the picomolar range (for review, see Refs 1, 2), thereby making unlikely the possibility that they act on human zona glomerulosa as true circulating hormones. However, ADM and PAMP are both synthesized and released by human adrenal medulla (for review, see Ref.2), so that they might act on the cortex in a paracrine manner, inasmuch as their intraglandular concentration could attain maximal values of about 10-8 mol/L (for review, see Ref.3). Andreis et al. (6) have pointed out that such local concentrations of ADM are compatible with the indirect stimulatory action of the peptide on aldosterone secretion (mediated by the enhanced catecholamine release by chromaffin cells), but not with its direct antimineralocorticoid effect. Preliminary results (not shown) indicate that, in contrast with ADM, PAMP does not affect basal catecholamine release by human adrenal medulla fragments, an expected finding in keeping with the contention that the catecholamine secretagogue action of ADM may be ascribed to its six-membered ring structure, of which PAMP is deprived (for review, see Ref.2). Moreover, our results show that such intraglandular concentrations are very compatible with the direct antimineralocorticoid effects of PAMP, occurring in a concentration range from 10-12 to 10-8 mol/L. Hence, it is reasonable to suggest that, of the two main preproadrenomedullin-derived peptides, PAMP is most likely to be considered the physiological inhibitory regulator of aldosterone secretion in humans.

In light of this hypothesis, our results obtained with ANG-II-responsive Conn’s adenomas may acquire extreme relevance. In fact, aldosteronoma dispersed cells not only exhibit secretory responses to ADM and PAMP qualitatively similar to those of normal human adrenocortical cells, but are significantly more sensitive to both peptides, and especially to PAMP, which is able to inhibit not only ANG-II-stimulated, but also basal aldosterone release. Studies are under way to ascertain whether this finding can be ascribed merely to the more elevated basal aldosterone secretion of tumor cells or if it can also depend on the presence of a higher number of specific receptors.

Schell et al. (11) have recently reviewed findings indicating that the blood concentrations of preproadrenomedullin-derived peptides are markedly increased in several pathological conditions where a resetting of fluid and electrolyte homeostasis is needed (e.g. essential hypertension, renal insufficiency, and chronic heart failure). Kato et al. (12) observed that this occurs also in primary hyperaldosteronism due to monolateral adrenal adenomas. Our dose-response curves indicate that the PAMP threshold concentrations able to depress the secretory activity of aldosteronoma cells are well below the blood levels attained by proadrenomedullin-derived peptides under such pathophysiological conditions (about 1–2 x 10-11 mol/L). Hence, in the case of Conn’s adenomas, PAMP could exert its mineralocorticoid antisecretagogue action, acting not only in a paracrine manner, but also as a true circulating hormone.

In conclusion, our present observations allow us to advance the working hypothesis that proadrenomedullin-derived peptides and especially PAMP may play a major role under pathophysiological conditions where an excess of aldosterone production has to be counteracted.

Received June 12, 1997.

Revised October 2, 1997.

Accepted October 6, 1997.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

  1. Richards AM, Nicholls MG, Lewis L, Lainchbury JG. 1996 Adrenomedullin. Clin Sci. 91:3–16.[Medline]
  2. Nussdorfer GG, Rossi GP, Mazzocchi G. 1997 Role of adrenomedullin and related peptides in the regulation of the hypothalamo-pituitary-adrenal axis. Peptides. 18:1079–1089
  3. Nussdorfer GG. 1996 Paracrine control of adrenal cortical function by medullary chromaffin cells. Pharmacol Rev. 48:495–530.[Medline]
  4. Yamaguchi T, Baba K, Doi Y, Yano K. 1995 Effect of adrenomedullin on aldosterone secretion by dispersed rat adrenal zona glomerulosa cells. Life Sci. 56:379–387.[Medline]
  5. Mazzocchi G, Rebuffat P, Gottardo G, Nussdorfer GG. 1996 Adrenomedullin and calcitonin gene-related peptide inhibit aldosterone secretion in rats, acting via a common receptor. Life Sci. 58:839–844.[CrossRef][Medline]
  6. Andreis PG, Neri G, Prayer-Galetti T, et al. 1997 Effect of adrenomedullin on the human adrenal glands: an in vitro study. J Clin Endocrinol Metab. 82:1167–1170.[Abstract/Free Full Text]
  7. Iwasaki H, Hirata Y, Iwashina M, Sato K, Marumo F. 1996 Specific binding sites for proadrenomedullin N-terminal 20 peptide (PAMP) in the rat. Endocrinology. 137:3045–3050.[Abstract]
  8. Andreis PG, Mazzocchi G, Rebuffat P, Nussdorfer GG. 1997 Effects of adrenomedullin and proadrenomedullin N-terminal 20 peptide on rat zona glomerulosa cells. Life Sci. 60:1693–1697.[CrossRef][Medline]
  9. Rossi GP, Albertin G, Belloni AS, et al. 1994 Gene expression, localization, and characterization of endothelin A and B receptors in the human adrenal cortex. J Clin Invest. 94:1226–1234.
  10. Mulrow PJ. 1992 Adrenal renin. Regulation and function. Front Neuroendocrinol. 13:47–60.[Medline]
  11. Schell DA, Vari RC, Samson WK. 1996 Adrenomedullin: a newly discovered hormone controlling fluid and electrolyte homeostasis. Trends Endocrinol Metab. 7:7–13.
  12. Kato J, Kitamura K, Kuwasako K, et al. 1995 Plasma adrenomedullin in patients with primary aldosteronism. Am J Hypertens. 8:997–1000.[CrossRef][Medline]



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