help button home button Endocrine Society JCEM
HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS
This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow Submit a related Letter to the Editor
Right arrow Alert me when this article is cited
Right arrow Alert me when eLetters are posted
Right arrow Alert me if a correction is posted
Right arrow Citation Map
Services
Right arrow Email this article to a friend
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Right arrow Request Copyright Permission
Citing Articles
Right arrow Citing Articles via HighWire
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Mazzocchi, G.
Right arrow Articles by Nussdorfer, G. G.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Mazzocchi, G.
Right arrow Articles by Nussdorfer, G. G.
The Journal of Clinical Endocrinology & Metabolism Vol. 89, No. 3 1277-1284
Copyright © 2004 by The Endocrine Society

Cholecystokinin (CCK) Stimulates Aldosterone Secretion from Human Adrenocortical Cells via CCK2 Receptors Coupled to the Adenylate Cyclase/Protein Kinase A Signaling Cascade

G. Mazzocchi, L. K. Malendowicz, F. Aragona, R. Spinazzi and G. G. Nussdorfer

Department of Human Anatomy and Physiology, Section of Anatomy, University of Padua (G.M., R.S., G.G.N.), I-35121 Padua, Italy; Department of Histology and Embryology, School of Medicine (L.K.M.), PL-60781 Poznan, Poland; and Department of Urology, Cannizzaro Regional Hospital (F.A.), I-95126 Catania, Italy

Address all correspondence and requests for reprints to: Prof. G. G. Nussdorfer, Department of Human Anatomy and Physiology, Section of Anatomy, Via Gabelli 65, I-35121 Padova, Italy. E-mail: gastone.nusdorfer{at}unipd.it.


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
Cholecystokinin (CCK) IS a regulatory peptide that acts via two receptor subtypes, CCK1-R and CCK2-R. RT-PCR demonstrated the expression of both CCK1-R and CCK2-R in the zona glomerulosa (ZG), but not zona fasciculata-reticularis cells of the human adrenal cortex. CCK and the CCK2-R agonist pentagastrin enhanced basal aldosterone secretion from ZG cells without affecting cortisol production from zona fasciculata-reticularis cells. The aldosterone response to CCK and pentagastrin was suppressed by a CCK2-R antagonist, but not by a CCK1-R antagonist. Pentagastrin evoked a sizeable cAMP, but not inositol triphosphate, response from ZG cells, whereas CCK plus CCK2-R antagonist was ineffective. The cAMP response to pentagastrin was abrogated by CCK2-R antagonist or the adenylate cyclase inhibitor SQ-22536, and the aldosterone response was abolished by both SQ-22536 and the protein kinase A inhibitor H-89. Both CCK and pentagastrin increased steroidogenic acute regulatory protein mRNA expression in ZG cells; the effect was abrogated by CCK2-R antagonist. We conclude that CCK exerts secretagogue action on human ZG cells, acting through CCK2-Rs coupled to the adenylate cyclase/protein kinase A signaling cascade, which, in turn, stimulates the expression of steroidogenic acute regulatory protein, the rate-limiting step of steroidogenesis.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
CHOLECYSTOKININ (CCK) IS a regulatory peptide that is widely distributed in the central nervous system and peripheral organs and is involved in the regulation of satiety, anxiety, and the neuroendocrine system (1, 2). CCK exerts its biological effects acting via two G protein-coupled receptors, referred to as CCK1-R and CCK2-R (formerly named CCK-A alimentary and CCK-B brain receptors) (3). From a phylogenetic point of view, CCK is believed to derive, along with gastrin, from a common ancestor, the CCK-like peptide (4), so it is understandable why the best characterized gastrin receptor is the CCK2-R (5). Among the several bioactive peptides derived from the posttranslational processing of progastrin, the five-amino acid residue pentagastrin appears to be the most effective agonist of the CCK2-R (4, 6).

Several lines of evidence indicate that CCK and its receptors are contained in the rat hypothalamo-pituitary-adrenal axis, the function of which they stimulate by enhancing ACTH and glucocorticoid secretion (7, 8, 9 ; and for review, see Refs. 1 and 2). Furthermore, it has been recently shown that CCK, acting via both CCK1-R and CCK2-R, enhance aldosterone secretion from dispersed rat zona glomerulosa (ZG) cells without affecting corticosterone production by zona fasciculata-reticularis (ZF/R) cells (10). In contrast with findings in the rat, studies dealing with CCK and human hypothalamo-pituitary-adrenal axis are scarce; CCK immunoreactivity has been detected in some substance P-positive nerve fibers of the adrenal cortex (11), and pentagastrin was found to raise ACTH and cortisol blood concentrations in healthy volunteers (12). Therefore, it seemed worthwhile to investigate whether human adrenocortical cells express CCK receptors, and whether CCK and pentagastrin are able to affect their secretory activity in vitro.


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

CCK and pentagastrin were purchased from Bachem (Bubbendorf, Switzerland), and the CCK1-R and CCK2-R antagonists PD-140,548 (3) and PD-135,158 (13) were obtained from Research Biochemical International (Natick, MA). Medium 199 was provided by Difco Laboratories (Detroit, MI). The adenylate cyclase inhibitor SQ-22536, the phospholipase C (PLC) inhibitor U-73122, the protein kinase A (PKA) inhibitor H-89, and the protein kinase C (PKC) inhibitor calphostin C (for references, see Ref. 14) were obtained from BIOMOL Research Laboratories (Milan, Italy). ACTH-(1–24), angiotensin II (Ang-II), human serum albumin, and all other laboratory reagents were purchased from Sigma-Aldrich Corp. (St. Louis, MO).

Preparation of adrenal specimens

Adrenal glands were obtained from 16 adult patients (from 35–62 yr old) undergoing unilateral nephrectomy/adrenalectomy for kidney cancer. Each patient gave written informed consent, and the study protocol was approved by the local ethics committees for human studies.

Portions of adrenal tails that do not contain medullary chromaffin tissue (15) were removed, placed in Krebs-Ringer bicarbonate buffer with 0.2% glucose at 4 C, and immediately carried to our laboratories. ZG was separated from inner ZF/R by stripping the capsule of adrenal tails and scrapping off adherent parenchymal tissue (16). Dispersed ZG and ZF/R cells were obtained by sequential enzymatic digestion and mechanical disaggregation (15). The reciprocal contamination of adrenocortical cell preparations, as evaluated by phase microscopy, was virtually absent. However, study of the expression of the CYP11B2 and CYP17 genes, encoding for P45011B2 (aldosterone synthase) and P450c17, indicated a low ZF/R cell contamination in ZG cell preparations. In fact, semiquantitative RT-PCR (see below) showed that ZF/R cell preparations expressed only CYP17 mRNA, whereas ZG cell preparations contained not only CYP11B2 mRNA, but also low levels of CYP17 mRNA (~10–15% of those present in dispersed ZF/R cells; Fig. 1Go).



View larger version (34K):
[in this window]
[in a new window]
  		FIG. 1. A, Ethidium bromide-stained 2% agarose gel showing cDNA amplified with human CYP11B2- and CYP17-specific primers from RNA of exemplary ZG (A) and ZF/R (B) cell preparations. The PCR program was 35 cycles of 95 C for 120 sec, 55 C for 60 sec, and 74 C for 90 sec. Lane 1 was loaded with 200 ng of a size marker (Marker VIII, Roche, Mannheim, Germany). The amplified fragments were of the expected size: 320 bp for CYP11B2, 751 bp for CYP17, and 100 bp for GAPDH. No amplification, with water instead of RNA, is shown as a negative control. B, Semiquantitative PCR of CYP11B2 and CYP17 expression in ZG and ZF/R cell preparations from adrenals 1–16. Expression is indicated by the ratio of amplicon density of target genes over that of GAPDH gene at 25, 30, and 35 PCR cycles. Bars are the mean ± SEM (n = 16).

 
Some of the dispersed cells obtained from each adrenal gland were frozen at -80 C, and some were immediately used for in vitro incubation experiments. In some experiments at the end of the incubation with CCK or pentagastrin, cells were recovered and frozen at -80 C.

RT-PCR

Total RNA was extracted from frozen dispersed cells and reverse transcribed to cDNA as described previously (17). For amplification of the resulting cDNA, 10 µl of the RT mixture were used. The sample volume was increased to 50 µl with a solution containing 50 mM KCl, 10 mM Tris (pH 8.3), 2 mM MgCl2, 0.1 µM up- and downstream primers, and 1 U Taq polymerase (AmpliTaq, PerkinElmer Corp./Cetus, Norwalk, CT). The primers and their thermal profile were selected with the software Primer-3, according to the methods described by Weinberg et al. (18) (CCK1-R and CCK2-R), Rossi et al. (19) (CYP11B2), Chamoux et al. (20) (CYP17), and Li et al. (21) [steroidogenic acute regulatory protein (StAR)]. In a thermal cycler (489 DNA TC; PerkinElmer Life Sciences, Milan, Italy), after a predenaturation step at 95 C for 120 sec, we used the PCR programs described in the legends of Figs. 1Go, 2Go, and 8Go. The primer sequences and predicted sizes of amplicons are shown in Table 1Go. An additional extension step at 72 C for 7 min was then carried out. To rule out the possibility of amplifying genomic DNA, in some experiments PCR was performed without prior RT of the RNA. As positive control, the expression of the housekeeping enzyme glyceraldehyde-3-phosphate dehydrogenase (GAPDH) was tested. Detection of the PCR amplification products was first performed by size fractionation on 2% agarose gel electrophoresis. After purification using the QIA-Quick PCR purification kit (Qiagen, Hilden, Germany), amplicons were identified by sequencing on an Alf sequencer (Pharmacia Biotech, Freiburg, Germany).



View larger version (44K):
[in this window]
[in a new window]
  		FIG. 2. A, Ethidium bromide-stained 2% agarose gel showing cDNA amplified with human CCK1-R- and CCK2-R-specific primers from RNA of ZG and ZF/R cell preparations of two (no. 1 and 2) exemplary human adrenal cortexes. The PCR program was 35 cycles of 94 C for 30 sec, 58 C for 30 sec, and 72 C for 90 sec. Lane 1 was loaded with 200 ng of a size marker (Marker VIII, Roche). The amplified fragments were of the expected sizes: 250 bp for CCK1-R, 260 bp for CCK2-R, and 585 bp for GAPDH. No amplification of PCR mixture, without prior RT of mRNAs, is shown as a negative control. B, Semiquantitative PCR of CCK1-R and CCK2-R expression in ZG and ZF/R cell preparations from adrenals 1–16. Expression is indicated by the ratio of amplicon density of target genes over that of GAPDH gene at 25, 30, and 35 PCR cycles. Bars are the mean ± SEM (n = 16).

 


View larger version (33K):
[in this window]
[in a new window]
  		FIG. 8. A, Ethidium bromide-stained 2% agarose gel showing cDNA amplified with human StAR-specific primers from RNA of exemplary control and 10-8 M CCK-treated ZG cell preparations. The PCR program was 30 cycles of 94 C for 45 sec, 64 C for 30 sec, and 72 C for 60 sec. Lane 1 was loaded with 200 ng of a size marker (Marker VIII, Roche). The amplified fragments were of the expected sizes: 974 bp for StAR and 585 bp for GAPDH. No amplification, with water instead of RNA, is shown as a negative control. B, Semiquantitative PCR of the effects of 10-8 M CCK (upper panel) and 10-8 M pentagastrin (PG; lower panel) on StAR expression in ZG cell preparations from adrenals 13–16. StAR responses were abolished by CCK2-R antagonist (CCK2-RA) and were unaffected by CCK1-R antagonist (CCK1-RA). Expression is indicated by the ratio of amplicon density of the target gene over that of the GAPDH gene at 20, 25, and 30 PCR cycles. Bars are the mean ± SEM (n = 4).

 

View this table:
[in this window]
[in a new window]
  		TABLE 1. RT-PCR primers and PCR products

 
Further experiments were carried out, according to Mazzocchi et al. (16), to provide a semiquantitative evaluation of CCK1-R, CCK2-R, CYP11B2, CYP17, and StAR expression. Briefly, amplification was performed for the number of PCR cycles indicated in Figs. 1Go, 2Go, and 8Go; gels were photographed on Polaroid films (Polaroid Italia, Varese, Italy); and amplicon band densities were measured with a digital image system (Leitz-Wetzlar, Wetzlar, Germany). The level of expression of the target genes was estimated by the ratio, at each number of PCR cycles, of their amplicon density to that of GAPDH, which was taken to equal 1.

Incubation experiments

Aliquots of dispersed cell suspensions (105 cells in 2 ml medium 199 and Krebs-Ringer bicarbonate buffer with 2% glucose containing 5 mg/ml human serum albumin) were incubated in duplicate as follows: 1) increasing concentrations of CCK or pentagastrin (from 10-12–10-5 M) and ACTH (10-8 M; ZG and ZF/R cell preparations from adrenals 1–4); 2) CCK1-R antagonist and/or CCK2-R antagonist (10-6 M) alone and in the presence of 10-8 M CCK or pentagastrin (ZG cell preparations from adrenals 5–8); 3) SQ-22536 (10-4 M), H-89 (10-5 M), U-73122 (10-5 M), or calphostin-C (10-5 M) alone and in the presence of 10-8 M pentagastrin (ZG cell preparations from adrenals 5–8); 4) SQ-22536 (10-4 M) or U-73122 (10-5 M) alone and in the presence of 10-8 M CCK plus 10-6 M CCK2-R antagonist, 10-8 M pentagastrin, 10-8 M ACTH, or 10-8 M Ang-II (ZG cell preparations from adrenals 9–12); and 5) CCK (10-8 M) plus CCK2-R antagonist (10-6 M) or pentagastrin (10-8 M) alone or in the presence of 10-6 M CCK1-R antagonist or 10-6 M CCK2-R antagonist, respectively (ZG cell preparations from adrenals 9–16). The incubation was carried out in a shaking bath at 37 C for 60 min (aldosterone secretion) or 10 min [cAMP and inositol triphosphate (IP3) production] in an atmosphere of 95% air-5% CO2. At the end of the experiments, the incubation tubes were centrifuged at 4 C at 100 xg for 10 min, and supernatants were stored at -80 C. ZG cells from adrenals 13–16 were collected, frozen, and stored at -80 C.

Steroid hormone assay

Aldosterone and cortisol were extracted from the incubation medium and purified by HPLC (22). Their concentrations were measured by RIA with commercial kits purchased from IRE-Sorin (Vercelli, Italy; ALDOCTK2 RIA kit: sensitivity, 15 pmol/liter; intra- and interassay coefficients of variation, 5.6% and 7.5%, respectively; cortisol RIA kit: sensitivity, 90 pmol/liter; intra- and interassay coefficients of variation, 6.3% and 8.0%, respectively).

cAMP and IP3 production

In the case of cAMP assay, the phosphodiesterase inhibitor 3-isobutyl-1-methylxantine (10-4 M) was added to prevent cAMP metabolism (10). 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 the cAMP concentration was determined following the protocol developed by Amersham Pharmacia Biotech (Little Chalfont, UK) for Biotrak TRK 432 (sensitivity, 1 pmol/liter; intra- and interassay coefficients of variation, 5.5% and 6.8%, respectively).

IP3 was extracted by the trichloroacetic acid method and purified by Amprep SAX-minicolumn chromatography (Amersham Pharmacia Biotech). The IP3 concentration was measured by RIA following the protocol developed by Amersham Pharmacia Biotech for Biotrak TRK 1000 (sensitivity, 2 pmol/liter; intra- and interassay coefficients of variation, 6.2% and 7.9%, respectively).

Statistics

Data were expressed as the mean ± SEM of the number of independent experiments indicated in the figures. Each experiment was performed with a cell suspension obtained from a single adrenal gland. Statistical analysis was carried out by ANOVA, followed by Duncan’s multiple range test.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Conventional and semiquantitative RT-PCR showed the expression of CCK1-R and CCK2-R mRNAs in ZG, but not ZF/R, cells of all 16 adrenal cortexes. GAPDH mRNA was detected in all ZG and ZF/R cell preparations (Fig. 2Go).

The viability of our dispersed cell preparations was demonstrated by their secretory response to ACTH (aldosterone response of ZG cells, 6.0/6.8-fold increase; cortisol response of ZF/R cells, 5.5/5.9-fold increase; Figs. 3Go and 4Go). CCK and pentagastrin concentration-dependently increased aldosterone secretion from ZG cells; their potency (half-maximal concentration) and efficacy (percent increase elicited by the maximal effective concentration, 10-8 M) were not significantly different [CCK vs. pentagastrin: half-maximal concentration, 1.2 ± 0.2 x 10-10 vs. 1.6 ± 0.3 x 10-6 M (P > 0.3, n = 4); efficacy, 216 ± 36 vs. 169 ± 28 (P > 0.3; n = 4); Fig. 3Go]. Neither CCK nor pentagastrin affected cortisol secretion from dispersed ZF/R cells (Fig. 4Go). CCK (10-8 M)- and pentagastrin (10-8 M)-induced aldosterone responses of ZG cells were suppressed by CCK2-R antagonist (10-6 M) and were unaffected by CCK1-R antagonist. Neither antagonist significantly altered basal aldosterone secretion (Fig. 5Go).



View larger version (19K):
[in this window]
[in a new window]
  		FIG. 3. Effects of CCK (upper panel) and pentagastrin (PG; lower panel) on basal aldosterone secretion from dispersed human ZG cells. Aldosterone response to 10-8 M ACTH is shown in the inset. Data are expressed per 106 cells and are the mean ± SEM of four separate experiments (adrenal cortexes 1–4). *, P < 0.05; **, P < 0.01 [vs. the respective baseline (B) value].

 


View larger version (18K):
[in this window]
[in a new window]
  		FIG. 4. Lack of effect of CCK (upper panel) and pentagastrin (PG; lower panel) on basal cortisol secretion from dispersed human ZF/R cells. The cortisol response to 10-8 M ACTH is shown in the inset. Data are expressed per 106 cells and are the mean ± SEM of four separate experiments (adrenal cortexes 1–4). **, P < 0.01 [vs. baseline (B) value].

 


View larger version (28K):
[in this window]
[in a new window]
  		FIG. 5. Effect of 10-6 M CCK1-R antagonist and/or CCK2-R antagonist (CCK1-RA and CCK2-RA, respectively) on basal and CCK-stimulated (10-8 M; upper panel) or pentagastrin-stimulated (PG; 10-8 M; lower panel) aldosterone secretion from dispersed human ZG cells. Data are expressed per 106 cells and are the mean ± SEM of four separate experiments (adrenal cortexes 5–8). **, P < 0.01 vs. the respective baseline value; A, P < 0.01 vs. the respective control value.

 
ZG cell preparations displayed marked cAMP and IP3 responses to ACTH and Ang-II, respectively (~6.0- and 5.5-fold increases); cAMP and IP3 responses were suppressed by the adenylate cyclase inhibitor SQ-22536 (10-4 M) and the PLC inhibitor U-73122 (10-5 M), respectively (Fig. 6Go). CCK (10-8 M) plus CCK2-R antagonist (10-6 M) did not evoke cAMP and IP3 responses from ZG cells. Conversely, pentagastrin (10-8 M) elicited a conspicuous cAMP response (3.0-fold increase), but not an IP3 response (Fig. 6Go). The cAMP response to pentagastrin was suppressed by either CCK2 antagonist (10-6 M) or SQ-22536 (10-4 M; Fig. 5Go). Both SQ-22536 (10-4 M) and the PKA inhibitor H-89 (10-5 M) abrogated the aldosterone response to pentagastrin (10-8 M), whereas U-73122 and the PKC inhibitor calphostin C were ineffective (Fig. 7Go). Basal aldosterone secretion was not affected by any of these inhibitors (Fig. 7Go).



View larger version (28K):
[in this window]
[in a new window]
  		FIG. 6. Effects of 10-8 M CCK plus 10-6 M CCK2-R antagonist (CCK2-RA) and 10-8 M pentagastrin (PG) on cAMP (upper panel) and IP3 (lower panel) release from dispersed human ZG cells. The cAMP response to 10-8 M ACTH and the IP3 response to 10-8 M Ang-II and their suppression by SQ-22536 (10-4 M) and U-73122 (10-5 M), respectively, are shown in the insets. The cAMP response to pentagastrin and ACTH is abolished by both CCK2-R antagonist (10-6 M) and SQ-22536 (10-4 M). Data are expressed per 106 cells and are the mean ± SEM of four separate experiments (adrenal cortexes 9–12). **, P < 0.01 vs. the respective baseline value; A, P < 0.01 vs. the respective control value.

 


View larger version (50K):
[in this window]
[in a new window]
  		FIG. 7. Effects of 10-4 M SQ-22536, 10-5 M H-89, 10-5 M U-73122, and 10-5 M calphostin C on basal and pentagastrin (PG; 10-8 M)-stimulated aldosterone secretion from dispersed human ZG cells. Data are expressed per 106 cells and are the mean ± SEM of four separate experiments (adrenal cortexes 9–12). **, P < 0.01 vs. the respective baseline value; A, P < 0.01 vs. the respective control value.

 
As expected, ZG cell preparations expressed the StAR gene. Semiquantitative PCR demonstrated that both CCK and pentagastrin evoked significant increases in StAR mRNA (4.8- and 3.1-fold, respectively); the effect was annulled by CCK2-R antagonist, but not CCK1-R antagonist (Fig. 8Go). Acute exposure to either CCK or pentagastrin did not significantly affect CYP11B2 expression (data not shown).


   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
Our study provides the first evidence that human adrenal ZG expresses the mRNAs of both CCK receptor subtypes, and that CCK enhances aldosterone secretion from dispersed ZG cells. These findings resemble those previously obtained in the rat (10). However, in contrast with this species, in humans the aldosterone secretagogue effect of CCK appears to be exclusively mediated by CCK2-R. In fact, 1) the CCK-stimulating action was abrogated by CCK2-R antagonist and was unaffected by CCK1-R antagonist; and 2) the potency and efficacy of CCK were not significantly different from those of the CCK2-R-selective agonist pentagastrin. The lack of CCK receptor gene expression in ZF/R and the absence of any glucocorticoid response of dispersed ZF/R cells to CCK are in keeping with earlier investigations carried out in the rat (7, 10). It should be noted that the absence of the secretory response of inner adrenocortical cells to CCK cannot be ascribed to their poor viability, inasmuch as they exhibit a normal response to their main agonist, ACTH.

It is currently accepted that G protein-coupled CCK receptors activate both adenylate cyclase- and PLC-dependent cascades (for references, see Ref. 3). However, our results strongly suggest that the CCK2-R-mediated aldosterone secretagogue effect of CCK and pentagastrin involves only the adenylate cyclase-PKA signaling mechanism. The following pieces of evidence support this contention: 1) the activation of CCK2-R, obtained by exposing dispersed ZG cells to either CCK plus CCK1-R antagonist or pentagastrin, markedly enhanced cAMP, but not IP3, production, and the cAMP response was suppressed by the CCK2-R antagonist; 2) the adenylate cyclase inhibitor SQ-22536, at a concentration able to abrogate the cAMP response to ACTH, abolished the aldosterone secretagogue effect of CCK and pentagastrin, and the same effect was elicited by the PKA inhibitor H-89; 3) SQ-22536 and H-89 per se did not evoke significant changes in basal aldosterone secretion over 60 min of static incubation, thereby ruling out the possibility that their effect was due to a nonspecific toxic lesion of the steroidogenic machinery; and 4) neither the PLC inhibitor U-73122, at a concentration that annulled the IP3 response to Ang-II, nor the PKC inhibitor calphostin C exerted any significant effect on the CCK- or pentagastrin-induced aldosterone response.

Compelling evidence indicates that StAR, by facilitating cholesterol transport to mitochondria (the rate-limiting step of steroidogenesis), is the main locus of action of several agonists, including ACTH (for review, see Refs. 23 and 24). It has been shown that ACTH increases StAR gene transcription in the target cells within 30–60 min (24), and our semiquantitative PCR data demonstrated that CCK and pentagastrin, which, like ACTH, activate the adenylate cyclase-dependent cascade, increased StAR mRNA in ZG cells within 60 min. This effect of CCK and pentagastrin is conceivably mediated by the CCK2-R, inasmuch as it was blocked by CCK2-R antagonist, but not by CCK1-R antagonist. ACTH is known to induce in adrenocortical cells the expression of several steroid hydroxylase genes, including CYP11B2 (for review, see Ref. 25). However, we did not observe any CCK- or pentagastrin-induced increase in CYP11B2 mRNA in ZG cells, thereby suggesting that this effect requires induction times longer than 60 min.

The physiological relevance of the CCK2-R-mediated direct aldosterone secretagogue effect of CCK remains to be ascertained. However, not only has the presence of CCK-positive nervous fibers been demonstrated in the human adrenal cortex (11), but also CCK, gastrin, and their precursors have been detected in pheochromocytomas (26) and in venous effluent of cat adrenals (27). It is well accepted the aldosterone secretion is finely tuned by several peptides, among which enkephalins, neuropeptide Y, vasoactive intestinal peptide, pituitary adenylate cyclase-activating polypeptide, and endothelins (for references, see Refs. 16 , 28 , and 29), and our findings suggest that CCK could be included in that group of regulatory peptides. In this connection, it should be noted that CCK is involved in the central regulation of satiety and that other peptides involved in the regulation of feeding (i.e. neuropeptide Y, orexins, and leptin) are known to control adrenocortical secretion acting at both the central (30, 31, 32) and the peripheral branch of the hypothalamo-pituitary-adrenal axis (33, 34, 35, 36, 37). It should be stressed that the multifactorial positive regulation of aldosterone secretion could be relevant in some forms of idiopathic hyperaldosteronism, including the rare variants associated with primary adrenal hyperplasia, where autonomous aldosterone secretion occurs in the absence of discrete adrenal adenomas (38, 39, 40). The possible involvement of CCK in the pathogenesis of such diseases surely awaits further investigations.

Another point needing to be addressed concerns the possible function of the CCK1-R that is expressed in the human ZG, but is not linked, as in the rat (10), with the CCK secretory effects. Is it a silent receptor? This possibility does not seem to be tenable, because in our hands CCK1-R antagonist did not potentiate the secretagogue action of CCK. Previous studies suggested that CCK, via the CCK1-R, enhances the proliferative activity of immature rat adrenocortical cells and thymocytes (41). According to the cell migration theory (for review, see Ref. 42), ZG in mammals is the cambium layer involved in adrenocortical cell renewal; hence, it is possible that CCK, via CCK1-R, plays a role in the regulation of adrenal growth.


   Footnotes
 
Abbreviations: Ang-II, Angiotensin II; CCK, cholecystokinin; GAPDH, glyceraldehyde-3-phosphate dehydrogenase; IP3, inositol triphosphate; PKA, protein kinase A; PKC, protein kinase C; PLC, phospholipase C; StAR, steroidogenic acute regulatory protein; ZF/R, zona fasciculata-reticularis; ZG, zona glomerulosa.

Received June 3, 2003.

Accepted November 17, 2003.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

  1. Crawley JN, Corwin RL 1994 Biological actions of cholecystokinin. Peptides 15:731–755[CrossRef][Medline]
  2. Liddle RA 1997 Cholecystokinin cells. Annu Rev Physiol 59:221–242[CrossRef][Medline]
  3. Noble F, Wank SA, Crawley JN, Bradwejn J, Seroogy KB, Hamon M, Roques BP 1999 International union of pharmacology. XXI. Structure, distribution, and functions of cholecystokinin receptors. Pharmacol Rev 51:745–781[Abstract/Free Full Text]
  4. Johnsen AH 1998 Phylogeny of the cholecystokinin/gastrin family. Front Neuroendocrinol 19:73–99[CrossRef][Medline]
  5. Kopin AS, McBride EW, Schaffer K, Beinborn M 2000 CCK receptor polymorphism: an illustration of emerging themes in pharmacogenomics. Trends Pharmacol Sci 21:346–353[CrossRef][Medline]
  6. Rehfeld JF, Johnsen AH 1994 Identification of gastrin component I as gastrin-71. The largest possible bioactive progastrin product. Eur J Biochem 223:765–773[Medline]
  7. Sander LD, Porter JR 1988 Influence of bombesin, CCK, secretin and CRF on corticosterone concentration in the rat. Peptides 9:316–320
  8. Kamilaris TC, Johnson EO, Calogero AE, Kalogeras KT, Bernardini R, Chrousos GP, Gold PW 1992 Cholecystokinin-octapeptide stimulates hypothalamic-pituitary-adrenal function in rats: role of corticotropin-releasing hormone. Endocrinology 130:1764–1774[Abstract/Free Full Text]
  9. Malendowicz LK, Jedrzejczak N, Macchi C, Markowska A, Tortorella C, Nussdorfer GG, Nowak M 1998 Effects of cholecystokinin on the pituitary-adrenal axis of rats with intact or regenerating adrenal gland. Biomed Res 19:303–308
  10. Malendowicz LK, Nowak M, Gottardo L, Tortorella C, Majchrzak M, Nussdorfer GG 2001 Cholecystokinin stimulates aldosterone secretion from dispersed rat zona glomerulosa cells, acting through cholecystokinin receptors 1 and 2 coupled with the adenylate cyclase-dependent cascade. Endocrinology 142:4251–4255[Abstract/Free Full Text]
  11. Heym C 1997 Immunocytochemical correlates of an extrapituitary adrenocortical regulation in man. Histol Histopathol 12:567–581[Medline]
  12. Abelson JL, Liberzon I 1999 Dose response of adrenocorticotropin and cortisol to the CCK-B agonist pentagastrin. Neuropsychopharmacology 21:485–494[CrossRef][Medline]
  13. Hughes J, Boden P, Costall B, Domeney A, Kelly E, Horwell DC, Hunter JC, Pinnock RD, Woodruff GN 1990 Development of a class of selective cholecystokinin type B receptor antagonists having potent anxiolytic activity. Proc Natl Acad Sci USA 87:6728–6732[Abstract/Free Full Text]
  14. Andreis PG, Markowska A, Champion HC, Mazzocchi G, Malendowicz LK, Nussdorfer GG 2000 Adrenomedullin enhances cell proliferation and deoxyribonucleic acid synthesis in rat adrenal zona glomerulosa: receptor subtype involved and signaling mechanism. Endocrinology 141:2098–2104[Abstract/Free Full Text]
  15. Andreis PG, Neri G, Prayer-Galetti T, Rossi GP, Gottardo G, Malendowicz LK, Nussdorfer GG 1997 Effects of adrenomedullin on the human adrenal glands: an in vitro study. J Clin Endocrinol Metab 82:1167–1170[Abstract/Free Full Text]
  16. Mazzocchi G, Malendowicz LK, Rebuffat P, Gottardo L, Nussdorfer GG 2002 Expression and function of vasoactive intestinal peptide, pituitary adenylate cyclase-activating polypeptide, and their receptors in the human adrenal gland. J Clin Endocrinol Metab 87:2575–2580[Abstract/Free Full Text]
  17. Mazzocchi G, Rossi GP, Neri G, Malendowicz LK, Albertin G, Nussdorfer GG 1998 11ß-Hydroxysteroid dehydrogenase expression and activity in the human adrenal cortex. FASEB J 12:1533–1539[Abstract/Free Full Text]
  18. Weinberg DS, Ruggeri B, Barber MT, Biswas S, Miknyocki S, Waldman SA 1997 Cholecystokinin A and B receptors are differentially expressed in normal pancreas and pancreatic adenocarcinoma. J Clin Invest 100:597–603[Medline]
  19. Rossi GP, Albertin G, Bova S, Belloni AS, Fallo F, Pagotto U, Trevisi L, Palù G, Pessina AC, Nussdorfer GG 1997 Autocrine-paracrine role of endothelin-1 in the regulation of aldosterone synthase expression and intracellular Ca2+ in human adrenocortical carcinoma NCI-H295 cells. Endocrinology 138:4421–4426[Abstract/Free Full Text]
  20. Chamoux E, Narcy A, Lehoux JG, Gallo-Payet N 2002 Fibronectin, laminin, and collagen IV as modulators of cell behavior during adrenal gland development in the human fetus. J Clin Endocrinol Metab 87:1819–1828[Abstract/Free Full Text]
  21. Li D, Sugawara T, Strauss III JF, Clark BJ, Stocco DM, Saenger P, Rogol A, Miller WL 1995 Role of steroidogenic acute regulatory protein in adrenal and gonadal steroidogenesis. Science 267:1828–1831[Abstract/Free Full Text]
  22. Neri G, Andreis PG, Prayer-Galetti T, Rossi GP, Malendowicz LK, Nussdorfer GG 1996 Pituitary adenylate cyclase activating peptide enhances aldosterone secretion of human adrenal gland: evidence for an indirect mechanism, probably involving the local release of catecholamines. J Clin Endocrinol Metab 81:169–273[Abstract]
  23. Stocco DM 2001 StAR protein and the regulation of steroid hormone biosynthesis. Annu Rev Physiol 63:193–213[CrossRef][Medline]
  24. Lehoux JG, Mathieu A, Lavigne P, Fleury A 2003 Adrenocorticotropin regulation of steroidogenic acute regulatory protein. Microsc Res Tech 61:288–299[CrossRef][Medline]
  25. Sewer MB, Watermann MR 2003 ACTH modulation of transcription factors responsible for steroid hydroxylase gene expression in the adrenal cortex. Microsc Res Tech 61:300–307[CrossRef][Medline]
  26. Bardram L, Hilsted L, Rehfeld JF 1989 Cholecystokinin, gastrin and their precursors in pheochromocytomas. Acta Endocrinol (Copenh) 120:479–484[Abstract/Free Full Text]
  27. Gaumann DM, Yaksh TL 1988 Effects of hemorrhage and opiate antagonists on adrenal release of neuropeptides in cats. Peptides 9:393–405[CrossRef][Medline]
  28. Nussdorfer GG 1996 Paracrine control of adrenal cortical function by medullary chromaffin cells. Pharmacol Rev 48:495–530[Medline]
  29. Nussdorfer GG, Rossi GP, Malendowicz LK, Mazzocchi G 1999 Autocrine-paracrine endothelin system in the physiology and pathology of steroid-secreting tissues. Pharmacol Rev 51:403–437[Abstract/Free Full Text]
  30. Spinedi E, Gaillard RC 1998 A regulatory loop between the hypothalamo-pituitary-adrenal (HPA) axis and circulating leptin: a physiological role of ACTH. Endocrinology 139:4016–4020[Abstract/Free Full Text]
  31. Krysiak R, Obuchowicz E, Herman ZS 1999 Interactions between the neuropeptide Y system and the hypothalamic-pituitary-adrenal axis. Eur J Endocrinol 140:130–136[Abstract]
  32. Malendowicz LK, Tortorella C, Nussdorfer GG 1999 Acute effects of orexins A and B on the rat pituitary-adrenocortical axis. Biomed Res 20:301–304
  33. Bornstein SR, Uhlmann K, Haidan A, Ehrhart-Bornstein M, Scherbaum WA 1997 Evidence for a novel peripheral action of leptin as a metabolic signal to adrenal gland. Leptin inhibits cortisol release directly. Diabetes 46:1235–1238[Abstract]
  34. Glasow A, Haidan A, Hilbers U, Breidert M, Gillespie J, Scherbaum WA, Chrousos GP, Bornstein SR 1998 Expression of Ob receptor in normal human adrenals differential regulation of adrenocortical and adrenomedullary function by leptin. J Clin Endocrinol Metab 83:4459–4466[Abstract/Free Full Text]
  35. Nussdorfer GG, Gottardo G 1998 Neuropeptide-Y family of peptides in the autocrine-paracrine regulation of adrenocortical function. Horm Metab Res 30:368–373[Medline]
  36. Renshaw D, Thomson LM, Carroll M, Kapas S, Hinson JP 2000 Actions of neuropeptide Y on the rat adrenal cortex. Endocrinology 141:169–173[Abstract/Free Full Text]
  37. Mazzocchi G, Malendowicz LK, Gottardo L, Aragona F, Nussdorfer GG 2001 Orexin A stimulates cortisol secretion from human adrenocortical cells through activation of the adenylate cyclase-dependent signaling cascade. J Clin Endocrinol Metab 86:778–782[Abstract/Free Full Text]
  38. Banks WA, Kastin AJ, Biglieri EG 1984 Primary adrenal hyperplasia: a new subset of primary aldosteronism. J Clin Endocrinol Metab 58:783–785[Abstract/Free Full Text]
  39. Biglieri EG, Irony I, Kater CE 1989 Identification and implication of new types of mineralocorticoid hypertension. J Steroid Biochem 32:199–204[CrossRef][Medline]
  40. Torpy DJ, Stratakis CA, Chrousos GP 1999 Hyper- and hypoaldosteronism. Vitam Horm 57:177–216[Medline]
  41. Malendowicz LK, Trejter M, De Caro R, Jedrzejczak N, Brelinska R, Markowska A, Nussdorfer GG, Nowak M 1999 Cholecystokinin, acting through the A receptor subtype, stimulates the proliferative activity of adrenocortical cells and thymocytes in the rat. Histol Histopathol 14:439–443[Medline]
  42. Nussdorfer GG 1986 Cytophysiology of the adrenal cortex. Int Rev Cytol 98:1–405[Medline]



This article has been cited by other articles:


Home page
 Physiol. Rev.Home page
M. Dufresne, C. Seva, and D. Fourmy
Cholecystokinin and gastrin receptors.
Physiol Rev, July 1, 2006; 86(3): 805 - 847.
[Abstract] [Full Text] [PDF]

Home page
 Pharmacol. Rev.Home page
R. Spinazzi, P. G. Andreis, G. P. Rossi, and G. G. Nussdorfer
Orexins in the regulation of the hypothalamic-pituitary-adrenal axis.
Pharmacol. Rev., March 1, 2006; 58(1): 46 - 57.
[Abstract] [Full Text] [PDF]

Home page
 J. Clin. Endocrinol. Metab.Home page
R. Spinazzi, M. Rucinski, G. Neri, L. K. Malendowicz, and G. G. Nussdorfer
Preproorexin and Orexin Receptors Are Expressed in Cortisol-Secreting Adrenocortical Adenomas, and Orexins Stimulate in Vitro Cortisol Secretion and Growth of Tumor Cells
J. Clin. Endocrinol. Metab., June 1, 2005; 90(6): 3544 - 3549.
[Abstract] [Full Text] [PDF]

Home page
 J. Clin. Endocrinol. Metab.Home page
G. Mazzocchi, P. Rebuffat, A. Ziolkowska, G. P. Rossi, L. K. Malendowicz, and G. G. Nussdorfer
G Protein Receptors 7 and 8 Are Expressed in Human Adrenocortical Cells, and Their Endogenous Ligands Neuropeptides B and W Enhance Cortisol Secretion by Activating Adenylate Cyclase- and Phospholipase C-Dependent Signaling Cascades
J. Clin. Endocrinol. Metab., June 1, 2005; 90(6): 3466 - 3471.
[Abstract] [Full Text] [PDF]

This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow Submit a related Letter to the Editor
Right arrow Alert me when this article is cited
Right arrow Alert me when eLetters are posted
Right arrow Alert me if a correction is posted
Right arrow Citation Map
Services
Right arrow Email this article to a friend
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Right arrow Request Copyright Permission
Citing Articles
Right arrow Citing Articles via HighWire
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Mazzocchi, G.
Right arrow Articles by Nussdorfer, G. G.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Mazzocchi, G.
Right arrow Articles by Nussdorfer, G. G.

HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS
Endocrinology Endocrine Reviews J. Clin. End. & Metab.
Molecular Endocrinology Recent Prog. Horm. Res. All Endocrine Journals