| HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
Articles |
Faculty of Pharmacy, Department of Pharmacology, Faculty of Medicine, Université de Montréal, Montréal, Québec H3C 3J7, Canada
Address all correspondence and requests for reprints to: Dr. A. De Léan, Department of Pharmacology, Faculty of Medicine, Université de Montréal, Case Postale 6128, Succursale Centre-Ville, Montréal H3C 3J7, Canada. E-mail: delean{at}ere.umontreal.ca
 |
Abstract |
|---|
 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
|
Introduction |
|---|
|
TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
To date, three receptors for PACAP have been cloned and are distinguished on a pharmacological basis by their relative affinity for PACAP and VIP and by the transduction pathways these peptides are able to activate. The type I receptor (PVR1) binds PACAP 100- to 1000-fold more potently than VIP and is coupled through G proteins to the activation of both adenylate cyclase (AC) and phospholipase C (PLC). Alternative splicing of this receptor may change the signal transduction characteristics by altering the structure of the third cytoplasmic loop putatively involved in G protein coupling. Type II receptors (PVR2 and PVR3) bind PACAP and VIP with approximately equal affinities. These receptors are coupled, probably through the G protein Gs, to the activation of AC (1, 2).
PACAP-like immunoreactivity and PACAP-containing nerve fibers have been demonstrated in the adrenal gland of several species (frog, mouse, hamster, rat, cow, and pig) (3), as well as in human pheochromocytomas (4). The occurrence of specific binding sites for PACAP has been shown in rat adrenal medulla (5) and on both adrenocortical and chromaffin cells of frog adrenal gland (6). In this later species, PACAP has been found to directly stimulate aldosterone and corticosterone secretion by dispersed, perifused adrenal cells. In rats and humans, PACAP stimulated aldosterone and corticosterone/cortisol secretion by adrenal slices, but not by dispersed adrenocortical cells, suggesting a rather indirect role of PACAP mediated by the chromaffin cells of the adrenal medulla (7, 8). In vivo studies have shown that PACAP increases the mean plasma cortisol concentration in conscious, functionally hypophysectomized calves (9).
The present study was designed to investigate the potential direct stimulatory effect of PACAP on aldosterone synthesis and secretion in mammalian species. Because responsiveness of a cell to PACAP will depend upon the type(s) of PVR expressed and the types of intracellular signaling pathway involved, it was tempting to compare the effects of PACAP in two different cell system models. The two models selected for our study are the bovine adrenal zona glomerulosa (ZG) cells in primary culture and the human adrenocortical carcinoma cell line H295R, which has been previously shown to retain functional control of aldosterone secretion by angiotensin II (AII) and atrial natriuretic peptide (ANP) (10, 11). The effects of PACAP and VIP on aldosterone, cAMP, and inositol(1, 4, 5)trisphosphate (Ins(1, 4, 5) P3) productions were studied in bovine and human adrenal cells. The effect of PACAP was compared with that of AII in both cell systems. Finally, the ability of ANP to inhibit PACAP-stimulated aldosterone synthesis was determined because this peptide is known as the physiological inhibitor of aldosterone synthesis and capable of counteracting the effects of most, if not all, secretagogues of this steroid hormone (12).
|
Materials and Methods |
|---|
|
TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
Cell culture
Primary culture of bovine adrenal ZG cells was performed as
described (13). Briefly, bovine adrenal glands were obtained from a
local slaughterhouse. The glands were cleaned of fat and a 0.5-mm layer
containing the capsule and the ZG was dissected with a scalpel. The
cells were dispersed in Ham’s F12 medium with 0.2% collagenase type
IA, 0.025% DNAse type I, and 0.25% BSA. Washed cells were resuspended
in Ham’s F12 medium supplemented with 10% horse serum, 2% FBS, 1%
streptomycin, 1% penicillin, and 2.5 mg/ml fungizone. The cell
suspension (106 cells/ml) was distributed in 1-ml fractions
in 24-well cluster plates for aldosterone and cAMP determinations and
in 5-ml fractions in 6-well plates for Ins(1, 4, 5)P3
determinations. Cell viability, as monitored by trypan blue exclusion,
was generally greater than 95%. Contamination of the ZG cell
preparations by zona fasciculata cells was less than 10% as attested
by morphological observation of the cells in the light microscope and
measurement of cortisol production by these cell preparations. The
cells were cultured in serum-containing medium for 4 days and starved
in serum-free medium for 48 h. The viability of the cells was not
affected during the 6 days of culture or by the various cell
treatments.
H295R cells were selected from the NCI-H295 cell line obtained from the American Type Culture Collection (ATCC, Rockville, MD) as previously reported (10). The cells were maintained in a 1:1 mixture of DMEM/F12 containing pyridoxine HCl, L-glutamine, and 15 mM HEPES and supplemented with insulin, transferrin, selenium (1% ITS+), 2.5% Nu-Serum, and antibiotics. Cells were grown in 75-cm2 flasks at 37 C under an atmosphere of 5% CO2-95% air until they reached confluence. Where aldosterone and cAMP production were studied, confluent cell monolayers were subcultured in 24-well cluster plates, and after 48 h, medium was replaced with fresh serum-free medium (DMEM/F12 containing 0.01% BSA). Cells were cultured further for 24 h, then rinsed and treated in the same medium. For Ins(1, 4, 5)P3 measurements, H295R cells were subcultured in 6-well plates.
Aldosterone determination
The cells were washed with their respective medium without
serum, and quadruplicate cell culture wells were stimulated for 3
h at 37 C with various concentrations of PACAP and VIP added to fresh
serum-free medium containing 0.01% BSA. At the end of the incubation,
the medium was removed quickly and frozen at -20 C until assayed for
aldosterone determination. Aldosterone was directly measured in cell
culture medium by a specific RIA as already described (14). The least
detectable concentration measured by the RIA was 5 fmol/ml.
cAMP determination
Bovine ZG cells and H295R cells were washed with their
respective serum-free medium and preincubated in 0.5 ml of the same
medium containing 0.01% BSA and 0.5 mM of the
phosphodiesterase inhibitor 3-isobutyl-1-methylxanthine (IBMX) for 20
min at 37 C. After the preincubation period, the cells were incubated
for 20 min at 37 C in the same medium containing various concentrations
of PACAP and VIP. At the end of the incubation, intracellular cAMP
content was extracted with 0.5 ml of 100% ethanol/10 mM
HCl. Cell extracts were evaporated to dryness in a Speedvac
concentrator (Savant) and resuspended in 50 µl of binding buffer.
cAMP was measured by a specific competitive binding assay using cAMP-dependent PKA from porcine heart as binding protein (15).The binding buffer consisted of 50 mM Tris-HCl pH 7.4 and 4 mM EDTA. Assays were conducted in 1.5-ml Eppendorff tubes and each contained 50 µl of either cAMP standard (0–100 pM) or cell extract, 50 µl of [3 H]-cAMP (approximately 50,000 cpm) and 100 µl of PKA (4 µg, diluted in binding buffer plus 0.1% BSA). The tubes were incubated on ice for 2 h. The reaction was terminated by adding 100 µl of 3.5% (wt/vol) charcoal in binding buffer plus 2% BSA. The tubes were briefly agitated, centrifugated (12,000 g, 5 min, 4 C) and 200 µl of the supernatant were taken for liquid scintillation counting. The lower limit of detection of this assay was 0.140 pmol cAMP per tube.
Inositol (1, 4, 5) trisphosphate determination
H295R cells and bovine ZG cells were washed twice and
preincubated 30 min at 37 C in incubation buffer (145 mM
NaCl, 5.6 mM KCl, 5.6 mM glucose, 0.01% BSA,
10 mM HEPES pH 7.4). After the preincubation period, the
cells were stimulated with PACAP and VIP in 1 ml of warmed incubation
buffer for 10 sec. The incubations were terminated by addition of 250
µl ice-cold perchloric acid (10%, vol/vol). The cells were scraped
with a rubber policeman and each well was washed with 250 µl 10%
perchloric acid. Samples were centrifugated (12,000 x
g, 5 min, 4 C), and the supernatants were neutralized with
1.5 M KOH containing 60 mM HEPES in the
presence of Universal Indicator.
Ins(1, 4, 5)P3 was quantified by a specific competitive binding assay using a crude microsomal preparation of bovine adrenal cortex (16). The binding assay was conducted in 1.5-ml Eppendorff tubes. Each incubation contained 25 µl of incubation buffer (100 mM Tris-HCl pH 9.0, 4 mM EDTA, 4 mM EGTA, 0.4% (wt/vol) BSA), 25 µl [3 H]-Ins(1, 4, 5)P3 (approximately 3000 cpm), 25 µl of standard Ins(1, 4, 5)P3 (0–100 pM) or cell extract and 25 µl of binding protein (0.5 mg). The tubes were agitated and incubated for 45 min on ice. Incubations were terminated by centrifugation (12,000 x g, 5 min, 4 C). The supernatant was removed by aspiration, and the pellet was dissolved in scintillation liquid and counted. The lower limit of detection of this assay was 0.200 pmol Ins(1, 4, 5)P3 per tube.
Data analysis
Dose-response curves for aldosterone and cAMP production were
analyzed with the ALLFIT for Windows1
program based on a four-parameter logistic equation to obtain estimates
of the ED50 (17). Statistical differences were tested by
ANOVA and Bonferroni multiple comparison t test after
checking for homogeneity of variance. Statistical significance levels
were set at P < 0.05 (significant) and
P < 0.01 (highly significant). All data presented are
representative of at least two separate experiments.
|
Results |
|---|
|
TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
). PACAP acts on
target cells by binding to cognate receptors. These receptors are
divided in two subtypes, each comprising multiple isoforms that can be
identified by their relative pharmacological profile towards PACAP38,
PACAP27, and VIP. As shown in Fig. 1A, all three peptides stimulated
aldosterone production with approximately the same efficacy
(ED50 = 0.52 ± 0.10, 0.14 ± 0.023, 0.70 ±
0.22 nM, respectively) in human tumorous H295R cells,
indicating that PACAP effect was mediated by the type II PACAP/VIP
receptor in these cells. Figure 1B shows that VIP had no stimulatory
effect in bovine ZG cells, whereas PACAP38 and PACAP27 both stimulated
aldosterone production with ED50 similar to those observed
in human cells (0.33 ± 0.066 and 0.55 ± 0.096
nM, respectively). Thus, PACAP seemed to act through type I
PACAP-specific receptor in bovine ZG cells. These results showed that
PACAP could directly stimulate aldosterone-producing cells in normal
bovine and human tumorous adrenal gland. However, they suggested a
species-dependent and/or cell type-dependent mode of action,
i.e. interaction with type I receptor in cow and type II
receptor in human tumor cells.
|
, PACAP38, PACAP27,
and VIP strongly stimulated cAMP production in a dose-dependent manner
in human H295R cells with ED50 of 0.35 ± 0.020,
0.20 ± 0.014, and 0.38 ± 0.021 nM,
respectively. In bovine ZG cells, only PACAP38 and PACAP27 increased
cellular cAMP content, although to a lesser extent than in human H295R
cells. The doses eliciting a half-maximum increase in cAMP production
were 0.12 ± 0.03 and 0.27 ± 0.042 nM,
respectively (Fig. 2B). VIP at a concentration of 100 nM
had no effect on AC activity in these cells. The ED50
values for cAMP production were in very good agreement with those
reported for aldosterone production (Fig. 1
). These results confirmed
the absence of type II PACAP/VIP receptor in bovine ZG cells.
|
). By contrast, in bovine ZG cells,
PACAP38 and PACAP27, at a concentration of 100 nM, elicited
a statistically significant 2.1- to 2.3-fold increase in Ins(1, 4, 5)P3 production (Fig. 3B). These results further
substantiated the differential expression of PACAP receptor type I and
type II in bovine ZG and human H295R tumorous cells, respectively.
|
). However, given
together, their effects were additive, and aldosterone production was
raised to 93.75 ± 4.50 fmol/well (P < 0.01).
This result was in accordance with the proposal that AII and PACAP
activated two distinct signaling pathways in human adrenocortical
carcinoma H295R cells: Ins(1, 4, 5)P3 and DAG for AII, and
cAMP for PACAP. The situation clearly was different in bovine ZG cells,
where AII was a more efficacious secretagogue than PACAP38 (7.06
± 0.76 and 4.38 ± 0.27 pmol/well, respectively), but most
importantly, the effects of AII and PACAP were not additive [8.23
± 0.27 pmol/well, not significant (NS)] in this species. This result
suggested that PACAP38 and AII shared, at least in part, a common
intracellular mechanism of action, i.e. the PLC-mediated
pathway, in stimulating aldosterone production in bovine ZG cells.
|
). In contrast, in bovine ZG
cells, ANP profoundly inhibited the maximal stimulatory effect of
PACAP38 (74%) and also shifted the ED50 of the curve to
higher concentration (0.52 ± 0.071 nM vs.
11.11 ± 6.46 nM) (Fig. 5B). The differential
inhibitory effect of ANP on PACAP-stimulated aldosterone production in
bovine ZG and human H295R cells could thus be the reflection of the
different transduction pathways mediating the effect of PACAP in the
two cell models.
|
|
Discussion |
|---|
|
TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
Comparison of the effect of PACAP38, PACAP27, and VIP on aldosterone
production in the two cell system models we used suggests that PACAP
interacts with a different receptor subtype in human adrenocortical
carcinoma cells and bovine ZG cells (Fig. 1). A comparative study of
the second messengers generated by PACAP and VIP in the two cell
systems further substantiates this hypothesis (Figs. 2 and 3).
Together, the results suggest that the direct steroidogenic action of
PACAP is mediated through PVR1 (type I PACAP-specific receptors)
coupled to both PLC and AC in bovine ZG cells but through PVR2 or PVR3
(type II PACAP/VIP receptors) coupled to AC in human H295R cells.
The rat PVR1 exists in six variant forms, a short form (PVR1s) and five splice variants having inserts in the third intracellular loop of the receptor, a domain believed to be important for interaction with G proteins (2). There are two distinct 28-amino acid inserts (termed hip and hop1), a 27-amino acid insert (hop2), and two combination inserts (termed hip-hop1 and hip-hop2). The presence of the hop insert alone has no influence on the potency of cAMP and Ins(1, 4, 5)P3 production. The hip insert alone abolishes coupling to PLC and alters potency of coupling to AC. The combination of the two inserts, hip-hop, gives an intermediate phenotype displaying slightly altered efficiency for AC stimulation and requiring high concentration (100 nM) for the stimulation of PLC. Likewise, four splice variants of the human PVR1 have been characterized, and the short and hop forms of the bovine PVR1 have been cloned (20, 21). In bovine ZG cells, PACAP stimulated cAMP production very potently (ED50 = 0.12–0.27 nM), but at a lower level than in human H295R cells, and was able to activate PLC. This suggests that PACAP receptors in these bovine adrenal cells are either of the PVR1s or the PVR1hop subtype if we refer to the rat nomenclature.
The effect of PACAP was compared with that of AII, the main physiological secretagogue of aldosterone. In human H295R cells, PACAP was as efficient as AII; both hormones induced a 3.6- to 4.9-fold increase in aldosterone production. In addition, their effects were additive in this species, with an 11.4-fold increase in steroid production with combined treatment. These results clearly suggest that PACAP and AII activate two distinct signaling pathways (cAMP and Ins(1, 4, 5)P3/Ca2+, respectively) in human adrenocortical carcinoma cells. The situation was strikingly different in bovine ZG cells, where AII was consistently more efficient than PACAP, with a 9.6-fold increase in aldosterone production as compared with a 5.9-fold increase induced by PACAP. Moreover, AII stimulation could not be enhanced by simultaneous treatment with PACAP. This situation is reminiscent of the cross-talk between GnRH and PACAP action in gonadotropes (1). In these cells, PACAP acts through PVR1 and stimulates both AC and PLC, whereas GnRH acts through a Gq-coupled receptor and stimulates only PLC. In both normal and clonal gonadotropes, the synergistic effect of PACAP on GnRH-stimulated gonadotropin release is only observed at lower (10 pM) concentrations, where PACAP would be expected to preferentially stimulate the cAMP/PKA pathway (PACAP is more potent in stimulating AC than PLC in these cells). At maximally effective concentration of both factors, GnRH inhibits PACAP-stimulated cAMP production, and synergy could no longer be observed because the two factors activate the same intracellular mechanism.
The last part of the present study concerns the effect of the natriuretic hormone ANP on PACAP-stimulated aldosterone secretion. As is true for all known secretagogues of aldosterone, ANP is able to inhibit the effect of PACAP in both human and bovine adrenocortical cells. However, the extent of this inhibition depends on the species and/or cell type concerned, being greater in cow than in human tumor cells. Barrett et al. (22) examined the inhibitory effect of ANP on aldosterone secretion stimulated by agonists that use either the Ca2+-phosphoinositide (AII) or the cAMP (ACTH) messenger system in bovine ZG cells. In these cells, ANP action is mediated by both a cyclic nucleotide-dependent and -independent pathway. Whereas the former is attributed to the activation of a cGS-PDE that hydrolyses the cAMP produced by ACTH activation (23), the latter operates through a yet unknown mechanism presumably acting downstream from the step of PLC activation of inositol phosphates production and intracellular calcium mobilization. The modest inhibitory effect of ANP on PACAP-stimulated aldosterone production in human H295R cells, where the neuropeptide activates the cAMP pathway exclusively, could be tentatively explained by analogy to ANP inhibition of ACTH-stimulated aldosterone production in bovine and rat ZG cells as described above. In addition, the lack of effect of ANP on PACAP ED50 on aldosterone production suggests that its inhibitory effect on cAMP intracellular accumulation is not altering a putative PACAP receptor reserve typically associated with a hyperbolic coupling of stimulus with cellular response (24). This is confirmed by our observation that PACAP ED50 on cAMP accumulation and on aldosterone production are superimposable, suggesting a linear coupling between second-messenger production and cellular response. These results are in sharp contrast with those obtained in bovine ZG cells, where ANP not only drastically inhibited PACAP-stimulated aldosterone production but also shifted to the right the concentration-response curve for PACAP. In this case, where PACAP stimulates both Ca2+-phosphoinositide and cAMP pathways, the cyclic nucleotide-independent pathway of ANP action seems to prevail over the cyclic nucleotide-dependent pathway. The rightward shift in the concentration-response curve produced by ANP could be explained in light of the model of functional antagonism developed by Van den Brink based on the concept of receptor reserve (24). This model predicts that, in a tissue or a cell type with a large receptor reserve, increasing the concentration of the antagonist will result initially in parallel shifts in the concentration-response curve to the agonist. However, when the receptor reserve is abolished, further increase in antagonist concentration will cause a diminution in the maximal response to the agonist. Because it is generally agreed that ANP does not alter inositol phosphates production or intracellular calcium mobilization (12), the nonlinear coupling step attenuated by ANP in bovine ZG cells, which also would be responsible for the apparent receptor reserve displayed, is probably located downstream from second messenger production.
In conclusion, investigation of the cross-talk between PACAP and ANP signaling pathways in two different cell system models bearing a different PACAP receptor subtype, which activates different signaling pathways, could improve our understanding of the intracellular mechanism of action of PACAP and ANP in regulating aldosterone biosynthesis.
|
Acknowledgments |
|---|
|
Footnotes |
|---|
2 Recipient of a PMAC-MRC research chair in Pharmacology sponsored by
Merck-Frosst Canada.
Received June 28, 1996.
|
References |
|---|
|
TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
This article has been cited by other articles:
 |
|
 |
|
  S. Valiante, M. Prisco, R. Sciarrillo, M. De Falco, A. Capaldo, F. Gay, P. Andreuccetti, V. Laforgia, and L. Varano Pituitary adenylate cyclase-activating polypeptide, vasoactive intestinal polypeptide and their receptors: distribution and involvement in the secretion of Podarcis sicula adrenal gland J. Endocrinol., February 1, 2008; 196(2): 291 - 303. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 G. Mazzocchi, L. K. Malendowicz, P. Rebuffat, L. Gottardo, and G. G. Nussdorfer Expression and Function of Vasoactive Intestinal Peptide, Pituitary Adenylate Cyclase-Activating Polypeptide, and Their Receptors in the Human Adrenal Gland J. Clin. Endocrinol. Metab., June 1, 2002; 87(6): 2575 - 2580. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 N. M. Sherwood, S. L. Krueckl, and J. E. McRory The Origin and Function of the Pituitary Adenylate Cyclase-Activating Polypeptide (PACAP)/Glucagon Superfamily Endocr. Rev., December 1, 2000; 21(6): 619 - 670. [Abstract] [Full Text]
|
|
|
|
 |
|
 L. BREAULT, L. YON, M. MONTERO, L. CHOUINARD, V. CONTESSE, C. DELARUE, A. FOURNIER, J.-G. LEHOUX, H. VAUDRY, and N. GALLO-PAYET Occurrence and Effect of PACAP in the Human Fetal Adrenal Gland Ann. N.Y. Acad. Sci., December 1, 2000; 921(1): 429 - 433. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 S. Gräs, C. Hedetoft, S. H. Pedersen, and J. Fahrenkrug Pituitary Adenylate Cyclase-Activating Peptide Stimulates Acute Progesterone Production in Rat Granulosa/Lutein Cells via Two Receptor Subtypes Biol Reprod, July 1, 2000; 63(1): 206 - 212. [Abstract] [Full Text]
|
|
|
|
 |
|
 D. Vaudry, B. J. Gonzalez, M. Basille, L. Yon, A. Fournier, and H. Vaudry Pituitary Adenylate Cyclase-Activating Polypeptide and Its Receptors: From Structure to Functions Pharmacol. Rev., June 1, 2000; 52(2): 269 - 324. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
M. RADZIKOWSKA, E. WASILEWSKA-DZIUBINSKA, and B. BARANOWSKA The Stimulatory Effect of VIP and PACAP on Adrenal Aldosterone Release Ann. N.Y. Acad. Sci., December 11, 1998; 865(1): 482 - 485. [Full Text] [PDF]
|
|
|
|
|
|
M. Ehrhart-Bornstein, J. P. Hinson, S. R. Bornstein, W. A. Scherbaum, and G. P. Vinson Intraadrenal Interactions in the Regulation of Adrenocortical Steroidogenesis Endocr. Rev., April 1, 1998; 19(2): 101 - 143. [Abstract] [Full Text]
|
|
| |||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
| 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 |
