Giuseppina Mazzocchi

Role of Adrenomedullin and Related Peptides in the Regulation of the Hypothalamo–Pituitary–Adrenal Axis

Adrenomedullin (ADM) is a hypotensive peptide, originally isolated from human pheochromocytomas, and then found to be widely distributed in the various body systems. ADM derives from preproadrenomedullin, a 185-amino acid residue prohormone, containing at its N-terminal a 20-amino acid sequence, named proadrenomedullin N-terminal 20 peptide (PAMP). ADM and PAMP immunoreactivities have been detected in the hypothalamo-pituitary-adrenal (HPA) axis of humans, rats, and pigs. Adrenal glands possess binding sites for both ADM and PAMP, the former being mainly of the subtype 1 of calcitonin gene-related peptide (CGRP) receptors. ADM exerts a direct inhibitory action on angiotensin II- or potassium-stimulated aldosterone secretion of zona glomerulosa cells. This effect is mediated by the CGRP1 receptor and its mechanism probably involves the blockade of Ca2+ influx. In contrast, ADM enhances aldosterone production by in situ perfused rat adrenals and human adrenal slices (containing medullary chromaffin cells), again through the activation of CGRP1 receptors. This aldosterone secretagogue effect of ADM is blocked by the beta-adrenoceptor antagonist l-alprenolol, thereby suggesting that it is indirectly mediated by the release of catecholamines by chromaffin cells. The effects of ADM on adrenal glucocorticoid release are doubtful and probably mediated by the increase in adrenal blood flow rate and the inhibition of ACTH release by pituitary corticotropes. The concentrations reached by ADM and PAMP in the blood rule out the possibility that they act on the HPA axis as circulating hormones. Conversely, their content in both adrenal and hypothalamo-pituitary complex is consistent with a paracrine mechanism of action, which may play a potentially important role in the regulation of fluid and electrolyte homeostasis.

Adrenomedullin and calcitonin gene-related peptide inhibit aldosterone secretion in rats, acting via a common receptor

Adrenomedullin (ADM) and calcitonin gene-related peptide (CGRP) did not affect either basal or ACTH-stimulated secretion of a1dosterone and corticosterone by dispersed rat capsular and inner adrenocortical cells, respectively. However, both peptides strongly depressed angiotensin-II (ANG- II)-stimulated a1dosterone production by capsular cells, the minimal effective concentration was 10(-7) M. The inhibitory effect of both ADM and CGRP was reversed by CGRP8-37, a specific CGRP1 receptor antagonist; a complete reversal was obtained with a CGRP8-37 concentration of 10(-6) M. Our findings indicate that ADM and CGRP specifically interfere with the intracellular mechanisms transducing the secretagogue signal of ANG-II, and suggest that the ADM effect is mediated by CGRP receptors

Adrenomedullin stimulates steroid secretion by the isolated perfused rat adrenal gland in situ: Comparison with calcitonin gene-related peptide effects

Adrenomedullin (ADM), a vasodilatatory peptide contained in adrenal medulla, was found to induce a dose-dependent increase in aldosterone (ALDO) and corticosterone (B) release by the in situ perfused rat adrenal gland, along with a rise in the flow rate of the perfusion medium. The minimal effective dose for ALDO response was three and two orders of magnitude less than those able to evoke B and medium flow rate responses. Calcitonin gene-related peptide (CGRP), another vasodilatatory peptide contained in adrenal medulla and showing a slight homology in its amino acid sequence with ADM, elicited similar effects. CGRP (8-37), a specific antagonist of CGRP1 receptors, annulled all the effects of both ADM and CGRP, whereas l-alprenolol, a beta-adrenoceptor antagonist, partially reversed only ALDO response to the peptides. In light of these findings the following conclusions are drawn: i) ADM and CGRP stimulate rat adrenals in vivo to release B by raising blood flow rate; ii) ADM and CGRP enhance ALDO secretion via an indirect mechanism probably requiring the release of catecholamines by medullary chromaffin cells; and iii) the effects of ADM and CGRP on the rat adrenal gland are mediated by a common receptor of the CGRP1 subtype.

Long-term effects of acth on rat adrenocortical cells: A coupled stereological and enzymological study

The effects of a long-term ACTH administration on the morphology of rat adrenocortical cells and the activity of some enzymes of steroid synthesis contained in them were examined by stereological and biochemical techniques. It was demonstrated that ACTH provokes time-dependent increases in the surface area per cell of smooth endoplasmic reticulum and mitochondrial cristae, which are tightly parallelled by enhancements in the activity per cell of delta 5-3 beta-hydroxysteroid dehydrogenase and 11 beta-hydroxylase, respectively.

Ghrelin enhances the growth of cultured human adrenal zona glomerulosa cells by exerting MAPK-mediated proliferogenic and antiapoptotic effects

Ghrelin is an endogenous ligand of the growth hormone secretagogue receptor (GHS-R), two subtypes of which have been identified and named GHS-R1a and GHS-R1b. Evidence has been provided that ghrelin and its receptors are expressed in the adrenal gland, and we have investigated the possible role of the ghrelin system in the functional regulation of the human adrenal cortex. Reverse transcription-polymerase chain reaction detected the expression of both subtypes of GHS-Rs exclusively in the zona glomerulosa (ZG). Ghrelin did not significantly affect either basal or agonist-stimulated aldosterone secretion from cultured ZG cells. In contrast, ghrelin raised proliferative activity and decreased apoptotic deletion rate of ZG cells, the maximal effective concentration being 10(-8) M. The growth effects of 10(-8) M ghrelin on cultured ZG cells were not affected by either the protein kinase (PK)A and PKC antagonists H-89 and calphostin-C or the mitogen-activated PK (MAPK) p38 antagonist SB-293580, but were abolished by both the tyrosine kinase (TK) and MAPK p42/p44 antagonists tyrphostin-23 (10(-5) M) and PD-98059 (10(-4) M), respectively. Ghrelin (10(-8) M) enhanced TK and MAPK p42/p44 activities of ZG cells. Preincubation with 10(-5) M tyrphostin-23 blocked the ghrelin-induced stimulation of both TK and MAPK p42/p44, while preincubation with 10(-4) M PD-98059 only annulled MAPK p42/p44 stimulation. Collectively, our findings allow us to conclude that ghrelin, acting via GHS-Rs exclusively located in the ZG, enhances the growth of human adrenal cortex, through a mechanism involving the activation of the TK-dependent MAPK p42/p44 cascade.

Effects of adrenomedullin and proadrenomedullin N-terminal 20 peptide on rat zona glomerulosa cells

Adrenomedullin (ADM) and proadrenomedullin N-terminal 20 peptide (PAMP) derive from a 185-amino acid prohormone, called preproadrenomedullin, which is highly expressed in rat adrenal medulla. ADM and PAMP did not affect either basal or ACTH-stimulated aldosterone secretion of dispersed rat zona glomerulosa cells In contrast, both peptides markedly suppressed angiotensin-II-stimulated aldosterone production, PAMP being much more effective than ADM (minimal effective concentration, 10(-10) M versus 10(-8) M. IC50, 2.0 +/- 0.17 x 10(-9) M versus 3.1 +/- 0.22 x 10(-8) M; P<0.01. Maximum inhibition, 80% versus 43%, respectively). The inhibitory effect of 10(-7) M ADM was completely reversed by the competitive antagonist of type 1 calcitonin gene-related peptide (CGRP) receptors CGRP(8-37) (10(-6) M), while that of 10(-7) M PAMP did not, thereby suggesting that this last peptide acts through specific receptors. Collectively, these findings may suggest that of the two main preproadrenomedullin derived peptides is PAMP which has probably to be considered a physiologic inhibitor of mineralocorticoid secretion in rats.

Blockade of Angiotensin II Type 1 Receptor and Not of Endothelin Receptor Prevents Hypertension and Cardiovascular Disease in Transgenic (mREN2)27 Rats via Adrenocortical Steroid–Independent Mechanisms

We investigated the role of angiotensin II (Ang II) and endothelin-1 (ET-1) in transgenic (mREN2)27 rats, a model of the monogenic renin-dependent form of severe hypertension and cardiovascular disease. Four-week-old heterozygous male transgenic (mREN2)27 rats (n=24) were matched according to body weight (BW) and blood pressure (BP) and randomly allocated to receive a placebo (group P), the mixed endothelin type A and B receptor antagonist bosentan (100 mg/kg BW PO, group B), the Ang II type 1-specific receptor antagonist irbesartan (50 mg/kg BW PO, group I), or the endothelin type A-selective antagonist BMS-182874 (52 mg/kg BW PO, group BMS). After 4 weeks of treatment, during which BW and BP were measured weekly, animals were euthanized, and the heart, left ventricle, right ventricle, adrenal gland, brain, and kidney were weighed. The plasma levels of adrenocortical steroids were measured by high-performance liquid chromatography. The tension responses of ET-free segments of the thoracic aorta to 5 x 10(-6) mmol/L phenylephrine, 60 mmol/L KCl, and cumulative doses of ET-1 were assessed. The density of ET-1 receptor subtypes in the aorta and vascular structural changes in the mesenteric arterioles (100 to 200 microm ID) were also measured with autoradiography and myography, respectively. Compared with all other groups, group I rats showed significantly (P<0.001) lower systolic BP (group I, 161+/-8 mm Hg; group P, 269+/-23 mm Hg; group B, 275+/-17 mm Hg; and group BMS, 254+/-21 mm Hg), left ventricular weight (2.28+/-0.15 versus 3. 71+/-0.26, 3.38+/-0.27, and 3.96+/-0.51 mg/g BW, respectively), tension responses to vasoconstrictors, and normalized media thickness of the mesenteric arterioles (22.3+/-0.6 versus 25.3+/-0.5, 25.5+/-0.7, and 24.1+/-1.5 microm, respectively). Compared with levels in group P (78+/-25 pmol/mL), plasma aldosterone levels were significantly decreased in group B (51+/-11 pmol/mL) and group I (40+/-16 pmol/mL). Thus, endogenous ET-1 and Ang II contribute to the regulation of aldosterone, but only Ang II is crucial for the development of hypertension and related target organ damage via the Ang II type 1 receptor. Endogenous Ang II does not appear to enhance cardiovascular production of ET-1 in this model of hypertension within the time span of our experiment.

11beta-hydroxysteroid dehydrogenase expression and activity in the human adrenal cortex.

Although oxidation of Cortisol or corticosterone by 11β‐hydroxysteroid dehydrogenase (11β‐HSD) represents the physiological mechanism conferring specificity for aldosterone on the mineralocorticoid receptor in mineralocorticoid target tissues, little attention has been paid until now to the expression and activity of this enzyme in human adrenals. We have shown that human adrenal cortex expresses 11β‐HSD type 2 (11β‐HSD2) gene, and found a marked 11β‐HSD2 activity in microsomal preparations obtained from slices of decapsulated normal human adrenal cortices. Under basal conditions, adrenal slices secreted, in addition to cortisol and corticosterone (B), sizeable amounts of cortisone and 11‐dehydrocorticosterone (DH‐B), the inactive forms to which the former glucocorticoids are converted by 11β‐HSD. Addition of the 11β‐HSD inhibitor glycyrrhetinic acid elicited a moderate rise in the production of cortisol and B and suppressed that of cortisone and DH‐B. ACTH and angiotensin II evoked a marked rise in the secretion of cortisol and B, but unexpectedly depressed the release of cortisone and DH‐B. ACTH also lowered the capacity of adrenal slices to convert [3H]cortisol to [3H]cortisone. This last effect of ACTH was concentration‐dependently abolished by both aminoglutethimide and cyanoketone, which blocks early steps of steroid synthesis, but not by metyrapone, an inhibitor of 11β‐hydroxylase. Collectively, these findings indicate that the human adrenal cortex possesses an active 11β‐HSD2 engaged in the inactivation of newly formed glucocorticoids. The activity of this enzyme is negatively modulated by the main agonists of glucocorticoid secretion through an indirect mechanism, probably involving the rise in the intra‐adrenal concentration of non‐11β‐hydroxylated steroid hormones.—Mazzocchi, G., Rossi, G. P., Neri, G., Malendowicz, L. K., Albertin, G., Nussdorfer, G. G. 11β‐Hydroxysteroid dehydrogenase expression and activity in the human adrenal cortex. FASEB J. 12, 1533–1539 (1998)

Short- and long-term effects of ACTH on the adrenal zona glomerulosa of the rat

SummaryShort-term ACTH treatment provoked a decrease in volume of the lipid-droplet compartment in rat zona glomerulosa cells, and a rise in plasma and intracellular concentrations of corticosterone and aldosterone. It enhanced activities of 3β-hydroxysteroid dehydrogenase (3βHSD), 11β-hydroxylase (11βOH) and 18-hydroxylase (18OH). Long-term ACTH administration produced a hypertrophy of the zona glomerulosa and its parenchymal cells, a result of the increase in volume of the smooth endoplasmic reticulum and the mitochondrial compartment. The surface area per cell of mitochondrial inner membranes increased; the tubular cristae were transformed into a homogeneous population of vesicles. The plasma and intracellular concentrations of corticosterone further increased, whereas those of aldosterone fell below basal levels (the “aldosterone-escape” phenomenon). The activities of 3βHSD and 11βOH were enhanced, that of 180H decreased. Therefore, ACTH stimulates zona glomerulosa growth and transforms parenchymal elements into zona fasciculata celltypes. Cyanoketone nullified acute ACTH effects on plasma and intracellular concentrations of corticosterone and aldosterone, but did not affect the activities of 11βOH and 18OH. Chronic ACTH treatment produced similar results, although 18OH activity was not suppressed. The mechanism underlying the “aldosterone-escape” phenomenon may thus involve a rise in the intracellular concentration of corticosterone, caused by the enhanced synthesis and activation of 3βHSD and 11βOH.

Immune-Endocrine Interactions in the Mammalian Adrenal Gland: Facts and Hypotheses

Several cytokines, which are the major mediators of the inflammatory responses, are well-known to stimulate the hypothalamopituitary corticotropin-releasing hormone (CRH)/adrenocorticotropic hormone (ACTH) system, thereby evoking secretory responses by the adrenal cortex. Many of these cytokines, including interleukin-1 (IL-1), IL-2, IL-6, tumor necrosis factor-alpha (TNF-alpha) and interferon-gamma (INF-gamma) are synthesized in the adrenal gland by both parenchymal cells and resident macrophages, and the release of some of them (e.g., IL-6 and TNF-alpha) is regulated by the main agonists of steroid hormone secretion (e.g., ACTH and angiotensin-II) and bacterial endotoxins. Adrenocortical and adrenomedullary cells are provided with specific receptors for IL-1, IL-2, and IL-6. IL-1 and TNF-alpha directly inhibit aldosterone secretion of zona glomerulosa cells, whereas IL-6 enhances it. IL-2, IL-3, IL-6, and INF-alpha are able to directly stimulate glucocorticoid production by zona fasciculata and zona reticularis cells, whereas IL-1 exerts an analogous effect through an indirect mechanism involving the stimulation of catecholamine release by chromaffin cells and/or the activation of the intramedullary CRH/ACTH system; again, TNF-alpha depresses glucocorticoid synthesis. IL-6 raises androgen secretion by inner adrenocortical layers. IL-1 enhances the proliferation of adrenocortical cells, and findings suggest that cytokines may control the apoptotic deletion of senescent zona reticularis cells. The relevance of the intraadrenal cytokine system in the fine-tuning of the secretion and growth of the adrenal cortex under normal conditions remains to be explored. However, indirect proof is available that local immune-endocrine interactions may play an important role in modulating adrenal responses to inflammatory and immune challenges and stresses.