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The Journal of Clinical Endocrinology & Metabolism Vol. 87, No. 6 2575-2580
Copyright © 2002 by The Endocrine Society

The Impact of the Human Genome on Endocrinology: Original Articles

Expression and Function of Vasoactive Intestinal Peptide, Pituitary Adenylate Cyclase-Activating Polypeptide, and Their Receptors in the Human Adrenal Gland

G. Mazzocchi, L. K. Malendowicz, P. Rebuffat, L. Gottardo and Gastone G. Nussdorfer

Department of Human Anatomy and Physiology, Section of Anatomy, University of Padua, I-35121 Padua, 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

VIP and pituitary adenylate cyclase-activating polypeptide (PACAP) are two regulatory peptides that possess remarkable amino acid sequence homology and act through common receptors, named PAC1, VPAC1, and VPAC2. PAC1 receptor is selective for PACAP, whereas VPAC1 and VPAC2 receptors bind both VIP and PACAP. We have investigated the expression and function of VIP, PACAP, and their receptors in the zona glomerulosa (ZG), zonae fasciculata and reticularis, and adrenal medulla (AM) of the human adrenal cortex. RT-PCR and RIA detected VIP and PACAP expression exclusively in AM cells. RT-PCR demonstrated the presence of PAC1 mRNA only in AM and of VPAC1 and VPAC2 mRNAs in both ZG and AM cells. VIP and PACAP concentration-dependently increased aldosterone and catecholamine secretion from cultured ZG and AM cells. The catecholamine response to both peptides was higher than the aldosterone response, and the secretagogue action of PACAP was more intense than that of VIP. The aldosterone response of cultured ZG cells to VIP or PACAP was unaffected by the PAC1 receptor antagonist PACAP-(6–38) (PAC1-A), but was significantly decreased by the VPAC1 receptor antagonist [Ac-His1,D-Phe2,Lys15,Arg16]VIP-(3–7),GH-releasing factor-(8–27)-NH2 (VPAC1-A). The catecholamine response of cultured AM cells to VIP was lowered by VPAC1-A and unaffected by PAC1-A; conversely, the catecholamine response to PACAP was reduced by both PAC1-A and VPAC1-A. Simultaneous exposure to both antagonists did not abolish the catecholamine response to PACAP. Collectively, our findings allow us to conclude that in human adrenals 1) VIP and PACAP biosynthesis exclusively occurs in AM cells; 2) ZG cells are provided with functional VPAC1 and VPAC2 receptors, whose activation by VIP or PACAP elicits a moderate aldosterone response; 3) AM cells possess PAC1, VPAC1, and VPAC2 receptors, whose activation evokes a marked catecholamine response; and 4) the catecholamine response to PACAP is more intense than that to VIP, because it is mediated by all subtypes of VIP/PACAP receptors.

VIP AND PITUITARY adenylate cyclase-activating polypeptide (PACAP) are two basic 28- and 38-amino acid C-terminally amidated peptides that were originally isolated from the gastrointestinal system (1) and hypothalamus, respectively (2). VIP and PACAP possess a remarkable amino acid sequence homology, which explains why they act through common G protein-coupled receptors (for review, see Refs. 3, 4, 5, 6, 7). At present, three main subtypes of VIP/PACAP receptors are recognized: PAC1, VPAC1, and VPAC2; their binding potency is: PAC1, PACAP>>>VIP; VPAC1, PACAP>=VIP; and VPAC2, PACAP=VIP.

Several lines of evidence indicate that VIP, PACAP, and their receptors are expressed in the mammalian adrenal glands (for review, see Refs. 7, 8, 9). The bulk of studies, carried out in rodents, showed that VIP and PACAP biosynthesis is restricted to adrenal medulla (AM), whereas VIP/PACAP receptor expression occurs in both zona glomerulosa (ZG) and AM. Investigations dealing with human adrenals are few, and their results can be summarized as follows. VIP immunoreactivity (-ir) and PACAP-ir were detected in adrenal homogenates (10, 11, 12). PAC1 receptor expression was found in fetal adrenomedullary chromaffin cells (13), and VPAC1/VPAC2 mRNAs were demonstrated in the adrenocortical carcinoma cell line NCI-H295 (14). VIP was shown to enhance aldosterone secretion from primary cultures of adrenocortical cells (15), and a sizeable secretory response to VIP and PACAP was observed in NCI-H295 cells (14, 16). However, freshly dispersed adrenocortical cells did not respond to PACAP (17). PACAP enhanced catecholamine release from adrenal slices containing AM (17).

It, therefore, seemed worthwhile to study the expression of VIP, PACAP, and their receptors in the cortex and medulla of human adrenals as well as to investigate the VIP/PACAP receptor subtypes involved in the adrenal secretory responses to both peptides.

Materials and Methods

Preparation of adrenal specimens

Adrenal glands were obtained from eight adult patients (40–45 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 committee for human studies.

Portions of adrenal tail and body, which do or do not contain and contain medullary chromaffin tissue (18), were removed, placed in Krebs-Ringer bicarbonate buffer with 0.2% glucose at 4 C, and immediately carried to our laboratory. ZG was separated from inner zonae fasciculata and reticularis (ZF/R) by stripping the capsule of adrenal tails and scrapping off adherent parenchymal tissue (19). AM was separated from the cortex of adrenal bodies under the dissecting microscope. Dispersed ZG, ZF/R, and AM cells were obtained by enzymatic digestion (18, 20). The reciprocal contamination of adrenal cell preparations, as evaluated by phase microscopy, was virtually absent. Part of the dispersed cells obtained from each adrenal gland was frozen at -80 C and used for gene expression studies, and part was immediately used for in vitro culture experiments.

RT-PCR

Total RNA was extracted from frozen dispersed cells and reverse transcribed to cDNA, as described previously (21). 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, Perkin-Elmer Corp./Cetus, Norwalk, CT). The primers and their thermal profile were selected with the software Primer-3, according to Haidan et al. (14) and Oka et al. (22). In a thermal cycler (Hybaid, Ashford, UK), after a predenaturation step at 94 C for 3 min, we used a denaturation step at 94 C for 60 sec, an annealing step at 57 C for 30 sec (VIP) or at 60 C for 60 sec [PACAP, VIP/PACAP receptor, and glyceraldehyde-3-phosphate dehydrogenase (GAPDH)], and an elongation step at 72 C for 90 sec for a total of 40 cycles. An additional extension step at 72 C for 7 min was then carried out. Primer sequences and predicted sizes of amplicons are shown in Table 1Go. To rule out the possibility of amplifying genomic DNA, one PCR was performed without prior RT of the RNA (data not shown). Detection of the PCR amplification products was first carried out by size fractionation on 2% agarose-gel electrophoresis. Then, after purification using the QIAQuick PCR purification kit (QIAGEN, Hilden, Germany), PCR products were identified by sequencing on an Alf sequencer (Pharmacia Biotech, Freiburg, Germany).


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  		Table 1. RT-PCR primers and PCR product

 
Further experiments were carried out to provide a semiquantitative evaluation of VIP/PACAP receptor expression. Amplification was performed for 20, 30, and 40 PCR cycles, 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 the housekeeping gene GAPDH, which was equal to 1.

VIP and PACAP RIA

Fresh adrenal specimens were immediately immersed in a cold mixture of water/ethanol/HCl (24.3:75.0:0.7, vol/vol/vol) and homogenized using a glass potter grinder. Homogenates were centrifuged at 4 C (13,000 x g for 10 min), and supernatants were collected and stored at -30 C (23). VIP and PACAP-38 concentrations were measured using kits provided by Phoenix Pharmaceuticals, Inc. (Belmont, CA): VIP (human, porcine, rat) RIA kit: sensitivity, 4.5 fmol/tube; cross-reactivity: VIP, 100%, PACAP-27, less than 0.03%, peptide histidine-methionine-27, less than 0.01%; and PACAP-38, less than 0.004%; PACAP-38 (human, ovine, rat) RIA kit: sensitivity, 6.5 fmol/tube; cross-reactivity: PACAP-38, 100%; PACAP-27, less than 0.01%; and VIP, 0. Intra- and interassay coefficients of variation were: VIP-ir, 5.9% and 7.6%; and PACAP-ir, 6.7% and 8.0%, respectively.

In vitro culture experiments

Freshly dispersed cells were suspended in Eagle’s MEM (Life Technologies, Inc., Paisley, UK) containing 4% FCS (Sigma, St. Louis, MO), 100 U/ml penicillin, 100 µg/liter streptomycin, and 50 µg/ml gentamicin. Cells were plated in 35-mm tissue culture dishes at a density of 105 cells/dish. Cells were cultured at 37 C in a humidified atmosphere of 95% air/5% CO2 and were employed after 24 h of culture.

Culture medium was replaced with fresh medium, which was collected after 60 min (ZG or ZF/R cell cultures) or 30 min (AM cell cultures) to evaluate basal hormonal secretion. New medium was then added, which contained the following peptides purchased from Peninsula Laboratories, Inc. (St. Helens, UK): VIP or PACAP-38 (from 10-10–10-6 M), the PAC1 receptor antagonist PACAP-(6–38) (PAC1-A), and/or the VPAC1 receptor antagonist [Ac-His1,D-Phe2,Lys15,Arg16-VIP-(3–7),GH-releasing factor-(8–27) (VPAC1-A; 10-6 M) alone or in the presence of 10-7 M VIP or PACAP-38. A further 60- or 30-min incubation at 37 C was then performed.

Steroid hormone and catecholamine assays

Aldosterone and cortisol were extracted from incubation medium and purified by HPLC as described previously (17). Their concentrations were measured by RIA with commercial kits purchased from IRE-Sorin (Vercelli, Italy): ALDO CTK2 RIA kit: sensitivity, 15 fmol/ml; intra- and interassay coefficients of variation, 5.8% and 7.3%, respectively; cortisol RIA kit: sensitivity, 90 fmol/ml; intra- and interassay coefficients of variation, 6.5% and 8.3%, respectively.

The catecholamine concentration in the incubation medium was measured by HPLC, using a reverse phase column and a glassy carbon electrochemical detector, as previously detailed (17). Catecholamines were about 50% norepinephrine and 50% epinephrine, and the sensitivity of the assay was 3 fmol/ml. Intra- and interassay coefficients of variation were 6.1% and 7.5%, respectively.

Values were expressed as the percent change from basal secretion, and data were expressed as the mean ± SEM of four independent experiments (four culture dishes obtained from each adrenal gland of four different patients). Statistical analysis was performed by ANOVA, followed by Duncan’s multiple range test.

Results

RT-PCR showed the expression of VIP and PACAP mRNAs only in AM cells, whereas GAPDH mRNA was detected in all adrenal preparations (Fig. 1AGo). Sizeable concentrations of VIP-ir and PACAP-ir were measured by RIA only in AM preparations (Table 2Go). PAC1 receptor mRNA was detected only in AM cells, whereas VPAC1 and VPAC2 receptor mRNAs were expressed in both ZG and AM preparations. ZF/R cells did not express VIP/PACAP receptors (Fig. 1BGo). Semiquantitative PCR showed that 1) the level of expression of VPAC1 and VPAC2 in the ZG cells and those of all three receptors in AM cells were nearly the same; and 2) VPAC1 and VPAC2 expression was more than 2-fold higher in AM than ZG cells (Fig. 1CGo).



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  		Figure 1. A and B, Ethidium bromide-stained 2% agarose gel showing cDNA amplified with human specific primers from RNA of ZG, ZF/R, and AM cells from two exemplary human adrenal cortexes (1 and 2). Forty PCR cycles were used as well as an additional extension step at 72 C for 7 min. Lanes 1 were loaded with 200 ng of a size marker (Marker VIII, Boehringer Ingelheim GmbH, Mannheim, Germany). No amplification with water instead of RNA is shown as a negative control. C, Semiquantitative PCR of VIP/PACAP receptor expression in human ZG and AM cells. Expression is indicated by the ratio of amplicon density of target genes over that of GAPDH gene. Bars are the mean ± SEM of four separate experiments (four ZG or AM cell preparations from adrenal glands of patients 5–8).

 

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  		Table 2. VIP and PACAP38 concentrations in adrenal glands of eight patients (fentomoles per mg protein)

 
VIP and PACAP concentration-dependently increased aldosterone secretion from cultured ZG cells; their potency (EC50) and efficacy (percent increase elicited by the maximal effective concentration, 10-7 M) were about the same (VIP vs. PACAP: EC50, 0.8 ± 0.3 x 10-8 vs. 1.1 ± 0.4 x 10-8 M; efficacy, 71 ± 9% vs. 77 ± 14%; P > 0.1; n = 4; Fig. 2AGo). Cortisol secretion from ZF/R cell cultures was not affected (data not shown). VIP and PACAP increased catecholamine release from cultured AM cells, but in this case PACAP was more effective than VIP [PACAP vs. VIP: maximal effective concentration, 10-8 vs. 10-7 M; EC50, 4.1 ± 1.0 x 10-9 vs. 1.2 ± 0.3 x 10-8 M (P < 0.05; n = 4); efficacy, 290 ± 50% vs. 192 ± 35% (P < 0.01; n = 4); Fig. 2BGo].



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  		Figure 2. Effect of VIP and PACAP on aldosterone (A) and catecholamine secretion (B) from cultured human ZG and AM cells, respectively. Data, expressed as percent change from baseline, are the mean ± SEM of four separate experiments (four ZG or AM cell culture dishes obtained from each adrenal gland of patients 1–4). *, P < 0.05; **, P < 0.01 [vs. baseline (B) value].

 
PAC1-A (10-6 M) did not alter either the aldosterone response of cultured ZG cells to VIP and PACAP (10-7 M; Fig. 3AGo) or the catecholamine response of cultured AM cells to VIP; in contrast, it lowered (by ~60%) the catecholamine response to PACAP (Fig. 3BGo). VPAC1-A (10-6 M) decreased both the aldosterone response of ZG cell cultures to VIP and PACAP (10-7 M; 67% and 60%, respectively) (Fig. 3AGo) and the catecholamine response of AM cell cultures to both peptides (63% and 37%, respectively); simultaneous exposure to PAC1-A and VPAC1-A did not annul the catecholamine response to PACAP (Fig. 3BGo).



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  		Figure 3. Effect of PAC1-A and/or VPAC1-A (10-6 M) on basal and VIP- or PACAP (10-7 M)-stimulated aldosterone (A) and catecholamine secretion (B) from cultured human ZG and AM cells, respectively. Data, expressed as the percent change from baseline, are the mean ± SEM of four separate experiments (four ZG or AM cell culture dishes obtained from each adrenal gland of patients 5–8). *, P < 0.05; **, P < 0.01 (vs. the respective baseline value). a, P < 0.05; A, P < 0.01 (vs. the respective control value).

 
Discussion

Our RT-PCR and RIA data confirm that in humans, as in other mammalian species (for references, see Refs. 7 and 9), VIP and PACAP biosynthesis occurs only in AM, in keeping with the abundance of these peptides in human pheochromocytomas (10, 11, 24, 25). The very low concentrations of VIP-ir and PACAP-ir measured in some ZG cell samples may be attributable to the islets and cords of chromaffin cells scattered in the ZG of mammalian adrenals (for review, see Refs. 8 and 26). VIP-ergic and PACAP-ergic fibers, largely of extrinsic (splanchnic nerve) origin, have been demonstrated by immunocytochemistry in the subcapsular cortex of several species (for references, see Ref. 27). However, they cannot account for our findings, because we have assayed the peptide concentrations in dispersed cells.

Several lines of evidence have shown that PAC1 receptors are exclusively located in adrenomedullary chromaffin cells of rats (28, 29), guinea pigs (30), and human fetuses (13), and accordingly PAC1 receptor mRNA was detected only in AM cells of adult human adrenals. Conversely, we demonstrated the expression of VPAC1 and VPAC2 receptors in both ZG and AM cells, a finding in accord with the autoradiographically reported distribution of [125I]VIP-binding sites in rat adrenal gland (31, 32, 33) Although Western blotting and immunocytochemical studies of VIP/PACAP receptors have not been performed, our functional findings clearly suggest that they are expressed not only as mRNAs, but also as proteins in human adrenals; in fact, VIP and PACAP are able to elicit secretory responses from ZG and AM cells, but not from ZF/R cells, where VIP/PACAP receptor expression is absent.

Previous studies did not find sizeable aldosterone responses of dispersed rat and human adrenocortical cells to VIP and PACAP (17, 34, 35). However, subsequent investigations showed that these peptides are able to stimulate aldosterone secretion from dispersed rat ZG cells (36, 37, 38) as well as from cultured human adrenocortical (15) and NCI-H295 cells (14, 16). In contrast, there is general consensus that VIP (39, 40, 41, 42, 43) and PACAP enhance catecholamine release from mammalian adrenomedullary cells (17, 44, 45, 46, 47).

In partial accordance with the above-reviewed studies, our present data provide evidence that VIP and PACAP stimulate aldosterone secretion from cultured human ZG cells through the activation of both VPAC1 and VPAC2 receptors and catecholamine release from cultured human AM cells via the activation of all three VIP/PACAP receptor subtypes. This conclusion is based on the following observations: 1) PAC1-A lowered the catecholamine, but not the aldosterone, response to the PAC1 receptor agonist PACAP; 2) VPAC1-A did not suppress, but only decreased, the aldosterone response to the VPAC1 receptor agonists VIP and PACAP, and when added together with PAC1-A did not abolish catecholamine response. In the lack of selective antagonists, these findings provide an indirect proof of the involvement of VPAC2 receptors in the secretagogue action of VIP and PACAP; and 3) the exposure to PAC1-A and/or VPAC1-A alone did not alter basal aldosterone or catecholamine production, thereby ruling out the possibility of their nonspecific toxic action on the secretory machinery of ZG and AM cells.

Our results also indicate that 1) the effects of VIP and PACAP on catecholamine secretion is more intense than that on aldosterone production, an observation in keeping with the higher expression of VIP/PACAP receptors in AM than ZG; and 2) the PACAP-evoked catecholamine response occurs through the activation of all subtypes of VIP/PACAP receptors and not, as previously reported, mainly via PAC1 receptors (13, 30, 48, 49, 50). It may be pointed out that our findings showing a direct aldosterone secretagogue action of VIP and PACAP on human ZG cells are in contrast with those obtained in an earlier study (17). We can tentatively explain this discrepancy by assuming that cultured vs. freshly dispersed adrenocortical cells were used; it is possible that 24-h culture allows ZG cells to recover the damage of VPAC1 and VPAC2 receptors ensuing from enzymatic digestion. Moreover, Neri et al. (17) used a mixture of dispersed outer and inner adrenocortical cells, where VIP/PACAP-unresponsive ZF/R cells prevailed (85% vs. 15%) over the responsive ZG ones.

The physiological relevance of our findings remains to be established, especially concerning the mild stimulatory effect of VIP and PACAP on ZG cells. According to Nussdorfer (8), it may be calculated that the adrenal content of VIP and PACAP, as measured by RIA in the present study, may give rise to a local concentration ranging between 2 and 5 x 10-8 M. This figure is compatible with an autocrine-paracrine mechanism of action of the two peptides, inasmuch as it is markedly higher than that able to elicit secretory responses from both ZG and AM cells. There is evidence that ß-adrenoceptor antagonists are able to hamper the aldosterone response to VIP and PACAP of adrenal quarters containing medullary chromaffin cells (17, 34, 35, 37, 51, 52). Hence, VIP and PACAP may modulate adrenocortical function, not only acting directly on ZG cells, but also by eliciting the release of catecholamines, which, in turn, stimulate aldosterone release.

Acknowledgments

Footnotes

Abbreviations: AM, Adrenal medulla; GAPDH, glyceraldehyde-3-phosphate dehydrogenase; ir, immunoreactivity; PACAP, pituitary adenylate cyclase-activating polypeptide; ZF/R, zonae fasciculata and reticularis; ZG, zona glomerulosa.

Received November 26, 2001.

Accepted March 1, 2002.

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