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Journal of Clinical Endocrinology & Metabolism , doi:10.1210/jc.2004-2132
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Right arrow Adrenal and Hypertension
The Journal of Clinical Endocrinology & Metabolism Vol. 90, No. 6 3466-3471
Copyright © 2005 by The Endocrine Society

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

G. Mazzocchi, P. Rebuffat, A. Ziolkowska, G. P. Rossi, L. K. Malendowicz and G. G. Nussdorfer

Departments of Human Anatomy and Physiology (G.M., P.R., G.G.N.) and Clinical and Experimental Medicine (G.P.R.), School of Medicine, University of Padua, I-35121 Padua, Italy; and Department of Histology and Embryology (A.Z., L.K.M.), Poznan School of Medicine, PL-60781 Poznan, Poland

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


   Abstract
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 Abstract
Introduction
 Patients and Methods
 Results
 Discussion
 References
 
Neuropeptides B and W (NPB and NPW) are regulatory peptides that act via two subtypes of G protein-coupled receptors, named GPR7 and GPR8. RT-PCR demonstrated the expression of these receptors in both zona glomerulosa and zona fasciculata-reticularis (ZF/R) cells of the human adrenal cortex. NPB and NPW did not affect aldosterone secretion from dispersed zona glomerulosa cells but enhanced cortisol production from ZF/R cells, NPB being more effective than NPW. NPB evoked sizable cAMP and inositol triphosphate responses from ZF/R cells, which were abrogated by the adenylate cyclase inhibitor SQ-22536 and the phospholipase C inhibitor U-73122, respectively. Cortisol response to NPB was lowered by either SQ-22536 and the protein kinase (PK) A inhibitor H-89 or U-73122 and the PKC inhibitor calphostin-C and abolished by the simultaneous exposure to H-89 and calphostin-C. NPW elicited only a rise in cAMP production from dispersed ZF/R cells, and its cortisol response was suppressed by both SQ-22536 and H-89. PreproNPB and preproNPW mRNAs were detected in human adrenal cortexes. We conclude that: 1) NPB and NPW exert a secretagogue action on human ZF/R cells, probably acting in an autocrine-paracrine manner; and 2) the effect of NPB is mediated by both the adenylate cyclase/PKA and the phospholipase C/PKC cascades, whereas that of NPW involves only the activation of the former signaling pathway.


   Introduction
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 Abstract
 Introduction
Patients and Methods
 Results
 Discussion
 References
 
NEUROPEPTIDES B AND W (NPB and NPW) are recently identified 29- and 30-amino-acid regulatory peptides, which are recognized to be endogenous ligands of G protein receptors (GPRs) 7 and 8 (1, 2). NPB and NPW bind and activate both receptors but with contrasting affinities: NPB, GPR7 > GPR8; and NPW, GPR8 > GPR7 (3, 4). GPR7 and GPR8 are encoded by two highly homologous genes, whose sequence is similar to that of somatostatin- and opioid-receptor genes. However, GPR7 and GPR8 do not bind somatostatin, and only GPR7 possesses a low affinity for nonselective opioid ligands (5). GPR7 and GPR8 are expressed in human brain, especially in the hypothalamic suprachiasmatic, supraoptic, dorsomedial, ventromedial, and paraventricular nuclei (5). GPR8 is absent in rodents, in which it is replaced by a GPR8-like receptor (6). NPW immunoreactivity has been detected in the rat hypothalamus and pituitary gland (7).

Evidence has been provided that GPR7, NPB, and NPW are involved in the central regulation of energy homeostasis and feeding behavior (8, 9). Moreover, although NPW did not affect ACTH release from dispersed rat anterior-pituitary cells, it was found to enhance the blood level of corticosterone in rats when administered intracerebroventricularly, thus suggesting an hypothalamic locus of action (stimulation of CRH and/or AVP release?) (10). Of interest, several regulatory peptides (e.g. opioids, neuromedins, neuropeptide Y, vasoactive intestinal polypeptide, pituitary adenylate cyclase-activating polypeptide, galanin, and neurotensin), which stimulate the central branch of the hypothalamic-pituitary-adrenal axis, are also able to act on the peripheral branch, i.e. adrenal cortex (for review, see Ref. 11), and accordingly NPB and NPW were found to increase proliferation and corticosterone secretion from cultured rat adrenocortical cells (12).

Therefore, it seemed worthwhile to investigate whether human adrenocortical cells express GPR7 and GPR8 mRNAs and whether NPB and NPW are able to affect their secretory activity in vitro.


   Patients and Methods
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 Abstract
 Introduction
 Patients and Methods
Results
 Discussion
 References
 
Reagents

NPB and NPW were purchased from Phoenix Pharmaceuticals (Belmont, CA). Medium 199 was provided by Difco Laboratories (Detroit, MI), and 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. 13) were obtained from BIOMOL Research Laboratories (Milan, Italy). ACTH(1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 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 male adult patients (from 44 to 65 yr old) undergoing nephrectomy/adrenalectomy for kidney cancer. Starting from 2 wk before surgery, patients were kept on a normal diet: only patients not requiring medications able to alter adrenal function were recruited. Each patient gave written informed consent, and the study protocol was approved by the local ethics committees for human studies.

Portions of adrenal tails, which contain no medullary chromaffin tissue (14), were removed, placed in Krebs-Ringer bicarbonate buffer with 0.2% glucose at 4 C, and immediately carried to our laboratories. Zona glomerulosa (ZG) was separated from inner zonae fasciculata and reticularis (ZF/R) by stripping the capsule of adrenal tails and scraping off adherent parenchymal tissue, and dispersed ZG and ZF/R cells were obtained by sequential enzymatic digestion and mechanical disaggregation (15). The contamination of ZG cell preparation by ZF/R cells was evaluated by measuring CYP17 mRNA expression (16): real time-PCR revealed that contamination did not exceed 15% (CYP17 relative expression: ZG cells, 0.185 ± 0.048 SD; and ZF/R cells, 1.331 ± 0.293 SD). Some of the dispersed cells obtained from each adrenal gland were frozen at –80 C and used for PCR assays, and some were immediately used to obtain freshly dispersed cells for in vitro incubation experiments.

RT-PCR

Total RNA extraction from frozen dispersed cells, its reverse transcription (RT) to cDNA, and the amplification of the resulting cDNA (thermal cycler 489 DNA TC; PerkinElmer Life Sciences, Milan, Italy) were performed as detailed earlier (16). To rule out the possibility of amplifying genomic DNA, in some experiments, PCR was carried out without prior reverse transcription of the RNA. As positive control, the expression of the housekeeping enzyme glyceraldehyde-3-phosphate dehydrogenase (GAPDH) was tested. Primer sequences, predicted sizes of amplicons, and PCR programs are indicated elsewhere (see the legends of Figs. 1Go, 6Go, and 7Go. Detection of the PCR amplification products was first performed by size fractionation on 2% agarose gel electrophoresis. After purification (QIA-Quick PCR purification kit; QIAGEN, Hilden, Germany), amplicons were identified by sequencing (Alf sequencer; Pharmacia Biotech, Freiburg, Germany).



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  		FIG. 1. Ethidium bromide-stained 2% agarose gel showing cDNA amplified with human GRP7-, GRP8-, and GAPDH-specific primers from RNA of exemplary ZG (a) and ZF/R cell (b) preparations. Primer sequences were as follows: 1) GRP7, sense, 5'-CTTGGAGAGCTGGAAACGAG-3' and antisense, 5'-GGACACAGATGGTGGACACG-3' (expected size of amplicon, 746 bp); 2) GRP8, sense, 5'-GCCACT-GCCGTTCCTCTAT-3' and antisense, 5'-GATGATGGGGGTGATGATGG-3' (expected size of amplicon, 898 bp); and 3) GAPDH, sense, 5'-CCCTTCATTGACCTCAACTA-3' and antisense, 5'-CCAGTGAG CTTCCCGTTCA-3' (expected size of amplicon, 585 bp). The PCR programs were: 1) GRP7 and GPR8, 34 cycles of 94 C for 60 sec, 60 C for 120 sec, and 72 C for 50 sec; and 2) GAPDH, 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, Mannheim, Germany). No amplification with water instead of RNA is shown as a negative control.

 


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  		FIG. 6. Top, Ethidium bromide-stained 2% agarose gel showing cDNA amplified with human StAR-, CYP17-, and CYP11B1-specific primers from RNA of exemplary control and 10–7 M NPB- or NPW-treated ZF/R cells. Primer sequences were as follows: 1) StAR, sense, 5'-GCAGCAGCAGCGGCGGCAGCAG-3' and antisense, 5'-ATCAGTGTGTGTACCAGT GCAC-3' (974 bp); 2) CYP17, sense, 5'-CCCATCTATTCTGTTCGTATGGGCAC-3' and antisense, 5'-GCCCCAAAGATGTCCCCTATGGTGGT-3' (751 bp); 3) CYP11B1, sense, 5'-CAGATGCAAGACTAGTTAATC-3' and antisense, 5'-ACATTGGTACAGCTTTTCCTC-3' (320 bp); and 4) GAPDH, sense, 5'-CTCTCTGCTCCTCCTGTTCG-3' and antisense, 5'-TGACTCCGACCTTCACCTTC-3' (100 bp). The PCR programs were: 1) StAR, 32 cycles of 94 C for 45 sec, 64 C for 30 sec, and 72 C for 60 sec; and 2) CYP17, CYP11B1, and GAPDH, 32 cycles of 95 C for 120 sec, 55 C for 60 sec, and 74 C for 90 sec. Lane 1 was loaded with Roche Marker VIII. No amplification with water instead of RNA is shown as a negative control. Bottom, Real-time PCR of the effects of 10–7 M NPB and NPW on StAR, CYP17, and CYP11B1 (adrenals 13–16). Bars, Mean ± SEM (n = 4). **, P < 0.01 vs. the respective control value.

 


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  		FIG. 7. Ethidium bromide-stained 2% agarose gel showing cDNA amplified with human ppNPB- (L7) and ppNPW (L8)-specific primers from an exemplary human adrenal cortex. Primer sequences were as follows: 1) ppNPB, sense, 5'-ACAGCTCCTACTCGGTG-3' and antisense, 5'-GCACCTTTGCAGGTTTGG-3' (189 bp); 2) ppNPW, sense, 5'-CTCCACTGCGCGCCCAAAC-3' and antisense, 5'-GCGTCTGCCACAGCTCCTG-3' (377 bp); and 3) GAPDH, see Fig. 1Go (585 bp). The PCR programs were: 1) ppNPB, 34 cycles of 94 C for 60 sec, 53 C for 90 sec, and 72 C for 30 sec; 2) ppNPW, 29 cycles of 94 C for 60 sec, 61 C for 90 sec, and 72 C for 30 sec; and 3) GAPDH, see Fig. 1Go. Lane 1 was loaded with Roche Marker VIII. No amplification of PCR mixture, without prior RT of mRNAs, is shown as negative control.

 
Real-time PCR

The relative expression of CYP11B1, CYP17, and steroidogenic acute regulatory protein (StAR) mRNAs in dispersed ZF/R cells was assayed in an I-Cycler iQ detection system (Bio-Rad Laboratories, Milan, Italy), using the primers indicated in Fig. 6Go and the following protocol: denaturation program (95 C for 3 min), 32 cycles of two steps of amplification (95 C for 15 sec and annealing for 30 sec), and melting curve (60–90 C with a heating rate of 0.5 C/10 sec). During the exponential phase, the fluorescence signal threshold was calculated, and the fraction number of PCR cycles required to reach the cycle threshold was determined. Cycle threshold values decreased linearly with increasing input target quantity and were used to calculate the relative mRNA expression. The specificity of amplification was tested at the end of each run by real-time PCR melting analysis, using the I-Cycler iQ software 3.0. All samples were amplified in duplicate and compared with the respective control. GAPDH was used as reference to normalize data.

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) NPB (from 10–12 to 10–6 M), NPW (from 10–12 to 10–6 M), ACTH (10–9 M) or Ang-II (10–8 M) (ZG and ZF/R cell preparations from adrenals 1–4); 2) 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 10–7 M NPB or NPW (ZF/R cell preparations from adrenals 5–8); 3) SQ-22536 or U-73122 alone and in the presence of 10–7 M NPB, 10–7 M NPW, 10–9 M ACTH, or 10–8 M Ang-II (ZF/R cell preparations from adrenals 9–12); and 4) H-89 plus calphostin-C alone and in the presence of 10–7 M NPB (ZF/R cell preparations from adrenals 9–12). The incubations were carried out in a shaking bath at 37 C for 60 min (steroid hormone 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 x g for 10 min, and supernatants were stored at –80 C.

Steroid hormone assay

Aldosterone and cortisol were extracted from the incubation media and purified by HPLC (17). Their concentrations were measured by RIA with commercial kits purchased from IRE-Sorin (Vercelli, Italy): ALDO-CTK2 RIA kit, sensitivity, 15 pmol/liter; intra- and interassay coefficients of variation (CVs), 5.5 and 7.3%, respectively; cortisol RIA kit, sensitivity, 90 pmol/liter; intra- and interassay CVs, 6.0 and 8.1%, 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 (18). 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 was determined following the protocol developed by Amersham Pharmacia Biotech (Little Chalfont, UK) for Biotrak TRK 432 (sensitivity, 1 pmol/liter; intra- and interassay CVs, 5.3 and 6.7%, 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 CVs, 6.2 and 8.0%. respectively).

Statistics

Data were expressed as the mean ± SEM of the number of independent experiments indicated in the figure legends. 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
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 Abstract
 Introduction
 Patients and Methods
 Results
Discussion
 References
 
RT-PCR showed the expression of GPR7 and GPR8 mRNAs in both ZG- and ZF/R-cell preparations of all 16 adrenal cortexes (Fig. 1Go). Semiquantitative PCR (15) findings did not evidence marked differences in the level of expression of either GPR7 or GPR8 expression between ZG and ZF/R cells (data not shown).

The viability of our freshly dispersed cell preparations was demonstrated by their secretory response to both ACTH and Ang-II (aldosterone response of ZG cells, ~9-and ~5-fold rises, respectively; cortisol response of ZF/R cells, ~10- and 2.7-fold rises, respectively) (Fig. 2Go). Neither NPB nor NPW affected aldosterone secretion from ZG cells (Fig. 2Go, upper panel). By contrast, NPB and NPW raised cortisol secretion from ZF/R cells, their maximal effective concentration (10–8/10–6 M) eliciting 2.5- and 2.0-fold increases, respectively (Fig. 2Go, lower panel).



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  		FIG. 2. Effects of NPB, NPW, ACTH (10–9 M), and Ang-II (10–8 M) on basal aldosterone secretion from dispersed human ZG cells (upper panel) and cortisol secretion from ZF/R cells (lower panel). Data are expressed per 106 cells and are the mean ± SEM of four separate experiments (adrenal cortexes 1–4). *, P < 0.05; and **, P < 0.01 vs. the respective baseline value.

 
ZF/R-cell preparations displayed marked cAMP and IP3 responses to ACTH and Ang-II, respectively. The cAMP and IP3 responses to both agonists were suppressed by the adenylate cyclase inhibitor SQ-22536 (10–4 M) and the PLC inhibitor U-73122 (10–5 M), respectively (Fig. 3Go). Both NPB and NPW elicited a cAMP response (~3-fold rise) (Fig. 3Go, upper panel), whereas only NPB evoked an IP3 response (~2-fold rise) (Fig. 3Go, lower panel).



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  		FIG. 3. Effects of NPB and NPW (10–7 M) on cAMP (upper panel) and IP3 (lower panel) release from dispersed human ZF/R cells. cAMP response to neuropeptides and ACTH (10–9 M) was suppressed by SQ-22536 (10–4 M) and IP3 response to neuropeptides and Ang-II (10–8 M) by U-73122 (10–5 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; b, P < 0.01 vs. the respective control value.

 
Cortisol response of ZF/R cells to NPB was partially suppressed by either SQ-22536 (10–4 M) and the PKA inhibitor H-89 (10–5 M) (~45% decrease) or U-73122 (10–5 M) and the PKC inhibitor calphostin-C (10–5 M) (~22% decrease), whereas cortisol response to NPW was abolished by SQ-22536 or H-89 and unaffected by U-73122 or calphostin-C (Fig. 4Go). When added together, H-89 and calphostin-C abrogated cortisol response to NPB (Fig. 5Go). Basal cortisol secretion from ZF/R cells was not affected by any of these inhibitors (Figs. 4Go and 5Go).



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  		FIG. 4. Effects of SQ-22536 (10–4 M), H-89 (10–5 M), U-73122 (10–5 M), and calphostin-C (10–5 M) on basal and 10–7 NPB- or NPW-stimulated cortisol secretion from dispersed human ZF/R cells. Data are expressed per 106 cells and are the mean ± SEM of four separate experiments (adrenal cortexes 5–8). *, P < 0.05; and **, P < 0.01 vs. the respective baseline value; a, P < 0.05; and b, P < 0.01 vs. the respective control value.

 


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  		FIG. 5. Effect of H-89 (10–5 M) plus calphostin-C (10–5 M) on basal and 10–7 NPB-stimulated cortisol secretion from dispersed human ZF/R 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; b, P < 0.01 vs. the respective control value.

 
As expected, ZF/R cell preparations expressed StAR, CYP17, and CYP11B1 genes. Real-time PCR showed that both NPB and NPW (10–7 M) evoked a significant increase in StAR mRNA (2.2- and 2.0-fold, respectively), without significantly affecting the expression of CYP17 and CYP11B1 (Fig. 6Go). Conventional RT-PCR detected the expression of prepro(pp)NPB (L7) and ppNPW (L8) mRNAs in all 16 adrenal cortexes (Fig. 7Go).


   Discussion
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 Abstract
 Introduction
 Patients and Methods
 Results
 Discussion
References
 
Our RT-PCR findings provide the first evidence that GPR7 and GPR8 genes are expressed in the human adrenal cortex, thereby confirming earlier studies carried out in the rat (1, 12). Moreover, because both receptors were found to be expressed in freshly dispersed ZG and ZF/R cells, the possibility that the stromal and vascular components of the gland can account for this result can be ruled out.

Despite the expression of GPR7 and GPR8 in both ZG and ZF/R cells, only the latter display a secretory response to NPB and NPW, thus raising question on the biological role of these receptors in ZG cells. According to the cell migration theory (for review, see Ref. 19), ZG in mammals is the cambium layer involved in adrenocortical cell renewal. Furthermore, previous investigations showed that NPB and NPW stimulate proliferation of cultured rat adrenocortical cells (12). Hence, the possible involvement of GPR7 and GPR8 located in ZG in the regulation of human adrenal growth under normal and perhaps pathological conditions, should be further addressed.

Both NPB and NPW elicit a marked cortisol response from dispersed ZF/R cells, but the effect of NPB is more intense than that of NPW at each concentration tested. NPB binds and activates GPR7 with greater affinity than GPR8 (3, 4). Although this has been demonstrated in cell systems other than adrenocortical one, it may be conceived that GPR7 plays the major role in the mediation of the NPB- and NPW-induced increase in cortisol secretion. The present findings are in contrast with those previously obtained in freshly dispersed rat adrenocortical cells, in which NPW at low concentrations raised basal aldosterone secretion, and both NPB and NPW enhanced ACTH-stimulated aldosterone production without affecting corticosterone output (12). At present, we can only tentatively suggest that interspecies differences may underlie these discrepancies between humans and rats, and in this connection we recall that rats do not possess true GPR8 (6).

Our study provides novel insight into the signaling mechanisms involved in the secretagogue action of NPB and NPW. Whereas both peptides activate adenylate cyclase/PKA-dependent cascade, only NPB activates PLC/PKC-dependent cascade. The following pieces of evidence support this contention: 1) NPB enhanced cAMP and IP3 production but NPW only cAMP production from ZF/R cells; 2) the adenylate cyclase inhibitor SQ-22536, at a concentration able to abrogate the cAMP response to ACTH, decreased cortisol response to NPB and abolished that to NPW, and the same effect was elicited by the PKA inhibitor H-89; 3) the PLC inhibitor U-73122, at a concentration that suppresses IP3 response to Ang-II, lowered cortisol response to NPB, but not to NPW, and the same effects were induced by the PKC inhibitor calphostin-C; 4) the simultaneous exposure to H-89 and calphostin-C annulled cortisol response to NPB; and 5) no signaling cascade inhibitor per se affected the basal cortisol secretion over 60 min of static incubation, thereby ruling out the possibility that their effect was due to a nonspecific toxic lesion of the ZF/R-cell steroidogenic machinery. The lack of selective antagonists of GPR7 and GPR8 did not allow us to conclusively ascertain which receptor subtype is coupled to the PLC-dependent cascade. However, due to the higher affinity of NPB for the GPR7, this subtype would seem to be the most plausible candidate. It could also be that NPB activates receptors other than GPR7 and GPR8, a hypothesis that needs to be tested in further studies.

It is commonly accepted that the main locus of action of several agonists, including ACTH and Ang-II, is StAR, a protein that facilitates cholesterol transport to mitochondria (the rate-limiting step of steroidogenesis) (for review, see Ref. 20). Evidence has been provided that ACTH raises StAR gene transcription in adrenocortical cells within 30–60 min (21). Our real-time PCR estimations showed that NPB and NPW, which, like ACTH, activate the adenylate cyclase-dependent cascade, increased StAR mRNA in ZF/R cells within 60 min. ACTH is also able to induce in adrenocortical cells the expression of several steroid hydroxylase genes, including CYP17 and CYP11B1 (for review, see Ref. 22). However, neither NPB nor NPW induced sizable increases in CYP17 and CYP11B1 mRNAs in ZF/R cells, which suggests that this effect might require induction times longer than 60 min.

The physiological relevance of the present findings remains to be ascertained. However, it is well demonstrated that in addition to the classic agonists ACTH and Ang-II (23), cortisol secretion is finely and variously tuned by several peptides, including enkephalins, endorphins, tachykinins, pancreatic polypeptide, galanin, gastric inhibitory polypeptide, and endothelins, many of which are locally synthesized in adrenal glands (for references, see Refs. 11 and 24, 25, 26). Thus, our present findings, coupled with the demonstration that both ppNPB and ppNPW mRNAs are expressed in adrenals, could suggest that NPB and NPW have to be included in that group of regulatory peptides, which modulate adrenocortical secretion probably acting in an autocrine-paracrine manner. Moreover, it is to be noted that NPB and NPW are involved in the central regulation of feeding (8, 9) and that other peptides playing a similar role (e.g. leptin and orexins) control glucocorticoid secretion acting at both the central (27, 28, 29) and the peripheral branch of the hypothalamus-pituitary-adrenal axis (29, 30, 31, 32, 33). It must be stressed that such multifactorial regulation of adrenal ZF/R secretion may be relevant in some dysregulations of glucocorticoid production, including some forms of ACTH-independent endogenous Cushing’s syndrome (for review, see Ref. 34). Accordingly, the possible involvement of NPB and NPW and GPR7 and GPR8 in the pathogenesis of such diseases awaits further investigations.


   Footnotes
 
First Published Online March 29, 2005

Abbreviations: Ang-II, Angiotensin-II; CV, coefficient of variation; GAPDH, glyceraldehyde-3-phosphate dehydrogenase; GPR, G protein receptor; IP3, inositol triphosphate; NPB, neuropeptide B; NPW, neuropeptide W; PKA, protein kinase A; PKC, protein kinase C; PLC, phospholipase C; pp, prepro; RT, reverse transcription; StAR, steroidogenic acute regulatory protein; ZF/R, zonae fasciculata and reticularis; ZG, zona glomerulosa.

Received October 29, 2004.

Accepted March 17, 2005.


   References
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 Abstract
 Introduction
 Patients and Methods
 Results
 Discussion
 References
 

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