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The Journal of Clinical Endocrinology & Metabolism Vol. 84, No. 2 632-635
Copyright © 1999 by The Endocrine Society

Original Studies

Cerebellin Enhances in Vitro Secretory Activity of Human Adrenal Gland

G. Mazzocchi, P. G. Andreis, R. De Caro, F. Aragona, L. Gottardo and G. G. Nussdorfer

Departments of Human Anatomy and Physiology (G.M., P.G.A., R.D.C., L.G., G.G.N.) and Urology (F.A.), University of Padua, I-35121 Padua, Italy

Address all correspondence and requests for reprints to: Prof. Gastone G. Nussdorfer, Department of Anatomy, Via Gabelli 65, I-35121 Padova, ltaly. E-mail: ggnanat{at}ipdunidx.unipd.it


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
Cerebellin is a 16-amino acid peptide, originally isolated from rat cerebellum, whose presence has been recently demonstrated in the human adrenal glands and especially in medullary chromaffin cells. Cerebellin concentration dependently increased basal catecholamine (norepinephrine and epinephrine) release by human adrenal slices, containing medullary chromaffin tissue, minimal and maximal effective concentrations being 10-9 and 10-7 mol/L. Cerebellin (10-7 mol/L) markedly enhanced cAMP release by adrenal slices, and the protein kinase A inhibitor H-89 (10-5 mol/L) blocked catecholamine response to cerebellin. Cerebellin did not affect basal steroid secretion of dispersed human adrenocortical cells, but it concentration dependently increased aldosterone and cortisol production by adrenal slices. Again minimal and maximal effective concentrations were 10-9 and 10-7 mol/L. Aldosterone and cortisol responses to 10-7 mol/L cerebellin was suppressed by both the ß-adrenoceptor antagonist l-alprenolol (10-6 mol/L) and H-89 (10-5 mol/L). Collectively, the present findings allow us to conclude that 1) cerebellin exerts a sizable secretagogue action on both cortex and medulla of human adrenals; 2) the peptide directly stimulates catecholamine release via the adenylate cyclase/protein kinase A-dependent signaling pathway; and 3) the mechanism underlying the adrenocortical stimulatory effect of cerebellin is indirect and probably involves the release of catecholamines, which in turn, acting in a paracrine manner, enhance steroid-hormone secretion.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
CEREBELLIN, a 16-amino acid peptide originally isolated from rat cerebellum (1), is widely distributed in the central nervous system, where it may exert a neuromodulatory function (2). The presence of cerebellin in the peripheral tissues is controversial (3), but cerebellin protein and mRNA have been recently detected in both normal and tumorous human adrenals (4). Immunocytochemistry showed that in the normal adrenals the presence of cerebellin is restricted to the medullary chromaffin tissue (4).

A large body of evidence indicates that several peptides expressed in and secreted by adrenomedullary chromaffin cells are able to control in a paracrine manner the secretory activity of the cortex, acting either directly or indirectly via the release of catecholamines, which in turn stimulate adrenocortical cells (for review, see Ref. 5). Hence, the present study was designed to investigate whether cerebellin affects in vitro adrenal function in humans, by using both dispersed adrenocortical cells and adrenal slices containing adrenomedullary tissue.


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

All chemicals were obtained from Sigma Chemical Co. (St. Louis, MO) with the following exceptions: cerebellin was supplied by Bachem (Bubendorf, Switzerland), H-89 from BIOMOL Research Laboratories, Inc. (Plymouth Meeting, PA), and medium 199 by DIFCO (Detroit, MI). Aldosterone and cortisol RIA kits were purchased from IRE-Sorin (Vercelli, Italy), and cAMP RIA from Amersham Labs (Amersham, UK).

Incubation experiments

Fragments of adrenal glands were obtained from adult patients (35–45 yr old) undergoing unilateral nephrectomy/adrenalectomy for kidney cancer. Only patients not requiring medications able to alter adrenal function were recruited. Starting from 2 weeks before surgery, patients were kept on a normal diet, i.e. unable to cause alterations of fluid and electrolyte balance, which may influence the secretory activity of adrenal zona glomerulosa. Portions of the head and tail of each adrenal, which are known to contain and respectively do not contain adrenal medulla (for references, see Ref. 6) were removed. The routine diagnostic examination of the remaining adrenal tissue did not show the presence of any histopathological alteration. The study protocol followed the local ethical committee guidelines for human studies.

Adrenal fragments were placed in Krebs-Ringer bicarbonate buffer with 0.2% glucose at 4 C and immediately carried out to our laboratory. Head fragments were cut into slices, always including the gland capsule and medulla; tail fragments, which are deprived of adrenal medulla, were employed to obtain dispersed adrenocortical cell preparations by collagenase digestion and mechanical disaggregation (6). Dispersed cells were actually a mixture of zona glomerulosa (aldosterone-secreting) and zona fasciculata-reticularis (cortisol-secreting) cells, the percentage of the two cell types (as evaluated by phase microscopy) being about 15% and 85%, respectively.

Dispersed adrenocortical cells and adrenal slices were placed in medium 199 and Krebs-Ringer bicarbonate buffer with 2% glucose, containing 5 mg/mL human serum albumin. The samples were incubated (3 x 105 cells or 4–5 mg/mL) as follows: 1) cerebellin (from 10-11 to 10-5 mol/L) and 2) 10-6 mol/L l-alprenolol or 10-5 mol/L H-89 in the presence or absence of 10-6 mol/L isoprenaline or 10-7 mol/L cerebellin (only adrenal slices). When cAMP production was assayed (see below), 10-4 mol/L of the phosphodiesterase inhibitor 3-isobutyl-1-methylxanthine was added to prevent cAMP metabolism. Incubation was carried out for 60 min (hormone production) or 10 min (cAMP production) in a shaking bath at 37 C in an atmosphere of 95% air-5% CO2. At the end of the experiments, the incubation tubes were centrifuged at 4 C, and media were collected and kept frozen at -80 C.

Hormonal and cAMP assays

Aldosterone and cortisol were extracted from supernatants and purified by HPLC (6); their concentrations were measured by RIA with the following commercial kits: 1) aldosterone CTK2 (sensitivity: 5 pg/mL; intra- and interassay variations, 5.7% and 7.4%, respectively); and 2) cortisol-RIA kit (sensitivity: 30 pg/mL intra- and interassay variations, 6.9% and 8.5%, respectively).

Catecholamine concentrations were assayed, without previous allumina purification and concentration, by HPLC with a reverse phase column and a glassy carbon electrochemical detector, as described previously (7). Intraassay and interassay variations were 7.0% and 8.5% for norepinephrine, and 6.9% and 8.3% for epinephrine, respectively.

cAMP was extracted by incubating the medium with 0.1 N HCl for 20 min at 4 C. The HCl extract was then neutralized and cAMP concentration determined using the acetylation protocol of the Amersham Labs Biotrak cAMP RIA system (sensitivity: 14 pg/mL; intraassay and interassay variations, 5.5% and 6.8%, respectively).

Statistics

Each incubation experiment was performed in triplicate or quadruplicate (three or four adrenals from three or four patients), and results were expressed as means ± SD. Their statistical comparison was done by ANOVA, followed by the multiple range test of Duncan.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Cerebellin concentration dependently raised basal catecholamine release by adrenal slices, minimal and maximal effective concentrations being 10-9 and 10-7 mol/L (Fig. 1Go). Norepinephrine response was more intense than that of epinephrine: although EC50 values were similar (1.07 ± 0.21 x 10-9 mol/L vs. 0.82 ± 0.10 x 10-9 mol/L), the rises evoked by the maximal effective concentration were 3.8-fold vs. 2.1-fold.



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  		Figure 1. Effect of cerebellin on catecholamine release by human adrenal slices containing medullary chromaffin tissue. Means ± SD of three separate experiments. +,P < 0.05; *, P < 0.01 vs. the respective baseline value (B).

 
Cerebellin (10-7 mol/L) increased cAMP release by adrenal slices (by about 14-fold) but not by dispersed adrenocortical cells (Fig. 2Go), and the catecholamine response to cerebellin (10-7 mol/L) was suppressed by 10-5 mol/L H-89 (Fig. 2Go).



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  		Figure 2. Effect of cerebellin on cAMP production by human adrenal slices and dispersed adrenocortical cells (left panel), and inhibitory action of H-89 (10-5 mol/L) on catecholamine response of human adrenal slices to cerebellin (right panel). Means ± SD of four separate experiments. *P < 0.01 vs. the respective baseline value (B); A, P < 0.01 vs. the respective control value.

 
Cerebellin did not affect basal aldosterone or cortisol production by dispersed adrenocortical cells (Fig. 3Go). In contrast, cerebellin increased in a concentration-dependent manner the secretion of both aldosterone and cortisol by adrenal slices, minimal and maximal effective concentrations being 10-9 and 10-7 mol/L (Fig. 4Go). EC50 values were 2.42 ± 0.29 x 10-9 mol/L and 1.48 ± 0.25 x 10-9 mol/L for aldosterone and cortisol, respectively; maximal effective concentration evoked about 3-fold rises in the output of both hormones. l-Alprenolol (10-6 mol/L) annulled the secretory response of adrenal slices not only to 10-6 mol/L isoprenaline (data not shown), but also to 10-7 mol/L cerebellin (Fig. 4Go). H-89 (10-5 mol/L) suppressed both aldosterone and cortisol response of adrenal slices to 10-7 mol/L cerebellin, without per se affecting basal secretion of both adrenal slices (Fig. 5Go) and dispersed adrenocortical cells (data not shown).



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  		Figure 3. Lack of effect of cerebellin on aldosterone and cortisol secretion of dispersed human adrenocortical cells. Means ± SD of three separate experiments.

 


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  		Figure 4. Effect of cerebellin on aldosterone and cortisol secretion of human adrenal slices, and its blockade by l-alprenolol (10-6 mol/L). Means ± SD of three separate experiments. +, P < 0.05; and *, P < 0.01 vs. the respective baseline value (B); AP < 0.01 vs. the respective control value.

 


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  		Figure 5. Inhibitory effect of H-89 (10-5 mol/L) on aldosterone and cortisol responses of human adrenal slices to cerebellin. Means ± SD of four separate experiments. *, P < 0.01 vs. the respective baseline value; A, P < 0.01 vs. the respective control value.

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
Our present findings show that cerebellin exerts a sizable stimulatory action on the release of catecholamines, and especially noradrenaline, by human adrenal chromaffin cells. This observation is in keeping with the reported exclusive localization of cerebellin in human adrenal medulla (4) and supports the contention that this peptide acts as a neuromodulator (2). Our results also suggest that cerebellin acts on the medullary chromaffin cells by activating the adenylate-cyclase/protein kinase A signaling cascade. In fact, cerebellin enhances cAMP release by adrenal slices containing medullary tissue, and catecholamine response to the maximal effective concentration of cerebellin is suppressed by H-89, a selective inhibitor of protein kinase A (8).

Cerebellin does not directly affect the secretory activity of dispersed human adrenocortical cells. However, it significantly enhances steroid hormone secretion by adrenal slices containing medullary tissue, thereby making likely the involvement of an indirect mechanism of action. Our results strongly suggest that the adrenocortical secretagogue effect of cerebellin may be mediated by the release of catecholamines. Compelling evidence indicates that the ß- adrenoceptor agonists are able to enhance adrenal steroidogenesis in mammals (for review, see Ref. 5) and recent findings showed that basal steroidogenic activity of bovine adrenocortical cells is increased 10-fold by coculture with chromaffin cells (9). Moreover, there is indication that other intramedullary regulatory peptides, e.g. pituitary adenylate cyclase-activating peptide (7) and adrenomedullin (6), stimulate steroid production by human adrenocortical cells through this indirect paracrine mechanism. The contention that cerebellin may be included in this group of regulatory peptides is supported by the present demonstration that the specific ß1-receptor antagonist l-alprenolol abolishes the secretory response of human adrenal slices, not only to the most potent ß- receptor agonist isoprenaline (10) but also to cerebellin. Moreover, H-89, in addition to suppressing catecholamine response to cerebellin, also annuls aldosterone and cortisol response of adrenal slices, but not of dispersed cells, to cerebellin. H-89 alone does not alter basal steroid secretion, thereby ruling out the possibility of its nonspecific toxic action on the steroidogenic machinery.

The physiological relevance of the stimulating effect of cerebellin on the human adrenal gland remains to be demonstrated. However, we wish to stress that, according to Nussdorfer (5), the reported content of 3 pmol/g cerebellin in normal human adrenals (4) could give rise, upon 30% release, to local concentrations of about 3 x 10-8 mol/L, which are well above the presently reported minimal effective concentration in vitro. It is conceivable that the catecholamine secretagogue effect of cerebellin may be very relevant in pheochromocytomas, where its concentration is about 30-times higher than in normal adrenals (4). The association of secreting pheochromocytomas with adrenocortical tumors and idiopathic hypercorticisms has been reported (5, 11, 12), and the herein described paracrine adrenocortical secretagogue action of cerebellin might at least in part explain the pathophysiological basis of these rare pathologies.

Received July 9, 1998.

Revised October 13, 1998.

Accepted October 19, 1998.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

  1. Slemmon JR, Blacher R, Danho W, Hempstead JL, Morgan JI. 1984 Isolation and sequencing of two cerebellum-specific peptides. Proc Natl Acad Sci USA. 81:6866–6870.[Abstract/Free Full Text]
  2. Burnet PWJ, Bretherton-Watt D, Ghatei MA, Bloom SR. 1988 Cerebellin-like peptide: tissue distribution in rat and guinea-pig and its release from rat cerebellum, hypothalamus and cerebellar synaptosomes in vitro. Neuroscience. 25:605–612.[CrossRef][Medline]
  3. Urade Y, Oberdick J, Molinar-Rode R, Morgan JI. 1991 Precerebellin is a cerebellum-specific protein with similarity to the globular domain of complement Clq B chain. Proc Natl Acad Sci USA. 88:1069–1073.[Abstract/Free Full Text]
  4. Satoh F, Takahashi K, Murakami O, et al. 1997 Cerebellin and cerebellin mRNA in the human brain, adrenal glands and the tumour tissues of adrenal tumour, ganglioneuroblastoma and neuroblastoma. J Endocrinol. 154:27–34.[Abstract/Free Full Text]
  5. Nussdorfer GG. 1996 Paracrine control of adrenal cortical function by medullary chromaffin cells. Pharmacol Rev. 48:495–530.[Medline]
  6. Neri G, Andreis PG, Prayer-Galetti T, Rossi GP, Malendowicz LK, Nussdorfer GG. 1996 Pituitary adenylate cyclase-activating peptide (PACAP) enhances aldosterone secretion of human adrenal gland: evidence for an indirect mechanism probably involving the local release of catecholamines. J Clin Endocrinol Metab. 81:169–173.[Abstract]
  7. Andreis PG, Neri G, Prayer-Galetti T, et al. 1997 Effects of adrenomedullin on the human adrenal glands: an in vitro study. J Clin Endocrinol Metab. 82:1167–1170.[Abstract/Free Full Text]
  8. Muroi M, Suzuki T. 1993 Role of protein kinase A in LPS-induced activation of NF-kappa B proteins of a mouse macrophage-like cell line, J774. Cell Signal. 5:289–298.[CrossRef][Medline]
  9. Haidan A, Bornstein SR, Glasow A, Uhlmann K, Lübke C, Ehrhart-Bornstein M. 1998 Basal steroidogenic activity of adrenocortical cells is increased 10-fold by coculture with chromaffin cells. Endocrinology. 139:772–780.[Abstract/Free Full Text]
  10. Lightly ERT, Walker SW, Bird IM, Williams BC. 1990 Subclassification of ß-adrenoceptors responsible for steroidogenesis in primary cultures of bovine adrenocortical zona fasciculata/reticularis cells. Br J Pharmacol. 99:709–712.[Medline]
  11. Gordon RD, Bachmann AW, Klem SA, et al. 1994 An association of primary aldosteronism and adrenaline-secreting phaeochromocytoma. Clin Exp Pharmacol Physiol. 21:219–222.[Medline]
  12. Tan GH, Carney JA, Grant CS, Young Jr WF. 1996 Coexistence of bilateral phaeochromocytoma and idiopathic hyperaldosteronism. Clin Endocrinol (Oxf). 44:603–609.[CrossRef][Medline]



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