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Original Studies |
Service of Endocrinology (E.C., L.B., N.G.-P.), Department of Biochemistry (J.-G.L.), Faculty of Medicine, University of Sherbrooke, Sherbrooke, Quebec, Canada J1H 5N4
Address all correspondence and requests for reprints to: Dr. Nicole Gallo-Payet, Service of Endocrinology, Department of Medicine, Faculty of Medicine, University of Sherbrooke, 3001 12th Avenue North, Sherbrooke, Quebec, Canada J1H 5N4. E-mail: n.gallo{at}courrier.usherb.ca
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Abstract |
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Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Several morphological evidences indicate that the human fetal adrenal gland is a very dynamic organ, in which proliferating cells located at the periphery migrate, differentiate, and finally undergo senescence in the central part of the gland. These indications stem from morphological examinations of picnotic figures (3). This cell loss is correlated with the dramatic reduction in weight of the adrenal gland after birth (for review, see Ref. 2). However, to date, there has been no detailed study of the process and control of programmed cell death (apoptosis) in the fetal adrenal gland.
It should be noted that the capacity for cell proliferation and cell migration persists in the adult gland. Indeed, according to previous studies, proliferating cells are located mainly in or near the zona glomerulosa (4), whereas apoptotic cells are predominant throughout the gland (5), in the zona reticularis (6), or in the zona glomerulosa (7, 8), highlighting the ongoing controversies with regard to the zonation or cell migration theories (8).
Among the factors known to modulate both mitotic activity and apoptosis is angiotensin II (Ang II), via its type 2 receptor (AT2). Inherent to the properties of the fetal adrenal gland, there are now several lines of evidence suggesting that activation of the AT2 receptor inhibits cell proliferation induced by growth factors and by stimulation of the Ang II type 1 receptor (AT1) (9, 10). A second role described for the AT2 receptor includes endocrine gland development (11, 12), neuronal differentiation (13, 14), and apoptosis (for review, see Ref. 15). Indeed, Ang II, through its AT2 receptor, was shown to initiate cell death and DNA fragmentation not only in neuronal cell types (16, 17), but also in endocrine (18, 19) and other tissues (20, 21). In the adult rat adrenal gland, apoptosis may be controlled by ACTH (protective effect) and Ang II (inducible effect) (22, 23).
In the adult adrenal gland, Ang II is one of the most important factors involved in the control of steroidogenesis. To date, all of the actions of Ang II in the adult adrenal gland have been associated with activation of the AT1 receptor, although 40% of AT2 receptors are also present in the zona glomerulosa (24, 25). A small proportion of Ang II receptors are present in zona fasciculata cells from bovine (26, 27) and human adrenal glands (24, 28), which are involved in the regulation of cortisol secretion. In contrast, previous studies from our laboratory have shown that fetal adrenal glands from the second trimester of gestation contain a large number of Ang II receptors, mostly of the AT2 subtype. The preferential distribution of the AT2 receptor in the fetus, compared to the adult, has been observed in several tissues, including rat (29, 30, 31) and human adrenal glands (24, 32, 33). Moreover, all of the components of the renin-angiotensin system are present in the meso- and metanephros and in placenta chorionic mesenchyme (33), suggesting that the AT2 receptor may be involved in fetal development.
When cells undergo apoptosis, morphological changes, such as membrane blebbing, nuclear chromatin condensation, and DNA laddering, are the most commonly used criteria to characterize apoptotic cell death (34, 35, 36). Several pieces of evidences implicate cysteine proteases called caspases as important proteolytic effectors that cleave key structural components and repair processes, resulting in organized cell disruption (37, 38). For example, actin-binding proteins and poly(ADP-ribose)polymerase (PARP protein) are known targets of caspases, such as caspase-3 (37, 38).
The aim of this study was to establish a link between the large number of AT2 receptors in fetal adrenal cells and proposed apoptotic activity in the center of the gland. We first verified the central localization of cells showing DNA degradation and demonstrated their characteristic apoptotic internucleosomal DNA fragmentation. We next studied the effect of Ang II on several of the characteristic events associated with apoptosis, such as formation of membrane blebs, fragmentation of chromatin, and PARP cleavage. Our results clearly indicate that the AT2 receptor is involved in the apoptotic process observed in the human fetal adrenal gland and could participate in the morphological changes occurring after birth, leading to involution of the fetal zone.
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Materials and Methods |
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The chemicals used in the present study were obtained from the following sources: deoxyribonuclease from Sigma (St. Louis, MO); Ang II from Bachem (Marina Delphen, CA); collagenase, Eagle’s MEM, and OPTI-MEM from Life Technologies, Inc.(Burlington, Canada); polyvinylidene difluoride membranes (Immobilon P) fromMillipore Corp. (Bedford, CA); anti-PARP antibody and anti-IgG antibody from Roche Molecular Biochemicals (Montreal, Canada); horseradish peroxidase-conjugated antirabbit IgG antibody and enhanced chemiluminescence detection system from Amersham Pharmacia Biotech (Oakville, Canada); TdT-fragEL kit from Calbiochem (Cambridge, MA); and Vecta-shield from Vector Laboratories, Inc. (Burlingame, CA). CGP 42112 was a gift from Dr. Marc de Gasparo (Novartis, Basel, Switzerland). PD123319 was obtained from RBI (Natik, MA). All other chemicals were of A grade purity.
Retrieval and preparation of glands
Fetal adrenal glands were obtained from fetuses aged 14–21 weeks (postfertilization) at the time of therapeutic abortion. Fetal ages were estimated by foot length and time after last menstruation, according to the method of Streeter et al. (39). The project was approved by the human subject review committee of our institution. After retrieval, glands were cleansed of fat and were either processed immediately for cellular preparation or quick-frozen in isopentane/dry ice and stored at -80 C until use.
Cell preparation
After fat removal, glands were processed as described previously (24). Small pieces of whole glands were incubated in Eagle’s MEM (supplemented with 100 U/ml penicillin and 100 µg/ml streptomycin) containing collagenase (2 mg/ml) and deoxyribonuclease (25 µg/ml) for 15 min at 37 C. Dissociation of cells was achieved by gentle aspiration in a sterile 10-ml pipette, then cells were filtered and centrifuged for 10 min at 100 x g. Cells were resuspended in OPTI-MEM medium supplemented with 2% FBS, 100 U/ml penicillin, 100 µg/ml streptomycin, and 0.5 µg/ml mycostatin. Cells were plated in 100-mm culture dishes at a density of 3 x 106 cells/dish for DNA fragmentation experiments or on plastic coverslips coated with poly-L-lysine at a density of 5 x 105 cells/dish for immunofluorescence studies. The cells were cultured at 37 C in a humidified atmosphere of 95% air-5% CO2. After a 24-h resting period, cells were incubated in culture medium in the absence or presence of Ang II and/or analogs for various time intervals.
Internucleosomal DNA fragmentation studies
Analysis of internucleosomal DNA fragmentation was performed as previously described (40, 41). In brief, after collecting both attached and floating cells, DNA was extracted with 0.5% Triton X-100, 10 mmol/L ethylenediamine tetraacetate, and 10 mmol/L Tris (pH 7.4). Lysates were three times phenol/chloroform extracted, ethanol precipitated, and analyzed on 1.5% agarose gel containing ethidium bromide. Separated DNA was visualized under UV light.
Western blotting of PARP
Samples from organotypic cultures of fetal adrenal glands were
solubilized according to the procedure of Lazebnick et al.
(42). Portions of adrenal glands were minced into small fragments and
homogenized in 3 vol Tris-HCl buffer containing 5 mmol/L
ethylenediamine tetraacetate, 5 mmol/L
ethyleneglycol-bis-(ß-aminoethyl
ether)-N,N,N',N'-tetraacetic
acid, 1 mmol/L dithiothreitol, 0.5 mmol/L phenylmethylsulfoxide, 10
µg/ml 4-(2-aminoethyl)-benzenesulfonyl fluoride, 5 µg/ml leupeptin,
10 µg/ml pepstatin, 10 µg/ml
N
-p-tosyl-L-lysine
chloromethyl ketone and 10 µg/ml
N-tosyl-L-phenylalanine chloromethyl
ketone. After 10–15 passes at 4 C, samples were aliquoted,
quick-frozen in isopentane/dry ice, and stored at -80 C until use. The
solubilized proteins were analyzed for content using a modified Lowry
assay. Whole cell protein samples (20 µg) were run on 12%
SDS-polyacrylamide gels (100 V, 45 min) and semidry transferred onto
polyvinylidene difluoride membranes at 0.55 A for 2 h. Blots were
blocked with 1% gelatin in Tris-buffered saline (pH 7.5), washed three
times with Tris-buffered saline-Tween-20 (0.05%) and probed with the
anti-PARP antibody (1:1000 dilution) overnight at 4 C. After five
washes, detection was accomplished using horseradish
peroxidase-conjugated antirabbit IgG antibody and enhanced
chemiluminescence detection system.
In situ DNA fragmentation studies
In situ DNA breakdown in whole glands was measured as described by Tilly et al. (43). Frozen glands were mounted in chunks and sliced into 5-µm sections using a cryostat at -20 C. The sections were thaw-mounted on gelatin-coated slides, placed for 30 min on a 37 C heating ramp to remove humidity, and processed for in situ experiments. The method used was similar to that of the TUNEL [terminal deoxynucleotidyl transferase-mediated deoxy (d)-UTP nick end labeling] method, but was performed with dCTP rather than dUTP nucleotides to label free 3'-ends of DNA. In brief, we used terminal deoxynucleotidyl transferase (TdT-fragEL Kit) to assemble biotinylated dCTP nucleotide at the 3'-extremities of double stranded DNA. Nuclei were detected after 3,3-diaminobenzidine revelation by an increased brown coloration in the case of strong DNA fragmentation.
Actin immunofluorescence
For immunofluorescence studies, cells were grown on plastic coverslips or petri dishes coated with poly-L-lysine (Starsted, St. Laurent, Canada). After a 24-h resting period, cells were treated for various time intervals with the appropriate stimuli. For visualization of microfilaments, cells were fixed for 20 min in 3% formaldehyde in PBS buffer, permeabilized for 10 min in PBS-0.1% Triton X-100, and incubated for 20 min at room temperature with 1 U rhodamine/phalloidin solution as described previously (44). After three washes, coverslips were mounted in Vectashield mounting medium and examined on a Nikon DM 400 microscope equipped for epifluorescence using the G-2A rhodamine filter (Nikon, Melville, NY).
Confocal microscopy
Cells were examined with a scanning confocal microscope (NORAN Instruments, Inc., Middleton, WI) equipped with a krypton/argon laser and coupled to an inverted microscope with a x40 oil immersion objective (Nikon). Specimens were excited at 568 nm, and the emitted rhodamine fluorescence was measured at wavelengths greater than 590 nm. Optical sections were collected at 0.25-µm intervals with a 10-µm pinhole aperture. Digitized images were obtained with 16 times line averaging and enhanced with Intervision software (NORAN) on a Silicon Graphics O2 workstation.
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Results |
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Patterns of internucleosomal DNA fragmentation were compared in
whole adrenal glands from fetuses between 14 and 20 weeks of gestation.
Figure 1
shows that before 16 weeks
gestation, there was no sign of apoptotic fragmentation, whereas it
increased in fetuses aged 16, 17, and 19 weeks. The localization of
apoptotic cells was examined by TUNEL. As shown in Fig. 2, the majority of the apoptotic cells in
frozen intact glands were observed in the central portion of the fetal
zone. No apoptotic nuclei were detected in the definitive or
transitional zones (Fig. 2A). Only occasional cells located in the
peripheral region surrounding the gland, i.e. around the
neocortex, were stained positively for apoptotic labeling. The number
of apoptotic cells increased from the periphery to the central portion
of the gland (Fig. 2, B and C), with maximal apoptotic labeling
observed in the center of the fetal zone, where the tissue exhibits a
low density of hypertropic cells (Fig. 2, D and E).
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We then examined whether apoptosis could be observed in cultured
cells (from adrenal glands at 17–20 weeks gestation) and whether Ang
II stimulation could influence this cell death. After 24 h of
culture, cells were stimulated for another 24 h without or with
100 nmol/L Ang II alone or in combination with analogs. Ang II slightly
increased internucleosomal DNA fragmentation compared to that in
control cells (Fig. 3). However, this
effect was more pronounced when cells were treated with 10 nmol/L
CGP42112, the AT2 receptor agonist, or with Ang
II and DUP753 (1 µmol/L), the AT1 receptor
antagonist.
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Cleavage of the DNA repair enzyme (PARP) into inactive fragments
by cysteine aspartate proteases (caspases) is known to contribute to
apoptosis (37, 38). Hence, we verified whether
AT2 receptor activation could affect PARP.
Western blot analysis using an anti-PARP antibody revealed that this
protein was cleaved in gland quarters from fetuses at 18 and 19 weeks
gestation cultured with 10 nmol/L CGP42112 for 24 h (Fig. 4). This protein was detected in the form
of a 116-kDa band protein corresponding to the native protein in
nonapoptotic gland quarters and in the form of a 90-kDa band protein
corresponding to the long fragment of PARP after proteolysis.
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When examined under phase contrast microscopy, 2-day treatment
with 10 nmol/L CGP42112 reduced the number of fetal cells attached to
the substratum (Fig. 5, B vs. A). In fact, cells had to be
cultured on poly-L-lysine-coated petri dishes, otherwise
all CGP42112-sensitive cells detached easily. Moreover, remaining cells
adopted a rounded shape, presented several blebs at the periphery, and
were observed exclusively on the steroidogenic fetal cells.
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and 6) (48). Cells from the
fetal zone (20–50 µm) have a well organized network of thin stress
fibers, crossing the entire surface of the cell, masking the nucleus. A
thin cortical ring of actin fibers was visible in most, but not all,
cells (Fig. 6A). A 1-min stimulation with
Ang II induced a rapid increase in actin fluorescence labeling at the
membrane, without any significant change in the stress fiber network,
compared to that in control cells (Fig. 6, B vs. A). A 2-h
incubation, on the other hand, induced a loss in the rigidity of actin
fibers and disappearance of actin labeling over the nucleus, making the
latter more clearly visible (Fig. 6C). When CGP42112 (10 nmol/L) was
used to stimulate only the AT2 receptor, the
effect was comparable to that observed with Ang II (Fig. 6, D and E),
except that the wavelling appearance of actin fibers was seen as early
as 1 min of incubation (Fig. 6D).
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, B and C) compared to control cells
(Fig. 7A). Examination of serial sections indicated that in control
cells, actin fibers are present over the entire cytoplasmic matrix
(Fig. 7, Ab and Ac), with a small ring of actin at the membrane
level (Fig. 7Ad). In apoptotic cells the intracellular actin network
had completely disappeared (Fig. 7, Ba to Bg and Ca to Cf), and
membrane blebbing was evident. Actin fluorescence was intense at the
cell membrane, with a predominant accumulation at the point of
initiation of bleb formation (Fig. 7, Bb to Bg and Cb to Cf). Figure 7C
shows three cells, all differentially affected by CGP42112. Although
the cell in the center (cell 1) exhibited cell blebbing, the cell on
the right (cell 2) remained insensitive to CGP42112 application,
whereas the cell on the left (cell 3) revealed a more precocious
effect, limited to a wavelling appearance of actin fibers.
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). After a 1-min
stimulation with Ang II, actin fibers became evident in cytoplasm and
decreased at the membrane (Fig. 8B). When CGP42112 was used as
stimulus, no evident modification in the actin network was visible,
either after 1 min or 2 h of incubation (Fig. 8C).
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Discussion |
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Programmed cell death or apoptosis is a physiological process essential in several aspects of tissue physiology not only for elimination of transformed or damaged cells, but also in the organization of developing tissues (34, 35, 36). Our results demonstrate that apoptotic DNA fragmentation appears after 16 weeks gestation and increases during the entire second trimester, suggesting that the apoptotic process is present and efficient early in gestation. Based on morphological criteria, some previous studies reviewed by Mesiano et al. (2) and supported by more recent data (49) indicated that apoptosis could be responsible for the involution of the fetal zone after birth. Using the TUNEL method to detect fragmented DNA in situ, we demonstrated that apoptosis occurs mainly in the center of the adrenal gland. This distribution is consistent with previous morphological observations on the process of adrenal gland development, whereby cells proliferate at the periphery, migrate centri-petally, then differentiate into fetal zone cells followed by apoptosis (2, 3).
The distribution of apoptotic cells in frozen glands corresponds to the pattern of AT2 expression previously described by our group (24). We thus examined whether Ang II could be involved in this apoptotic process. Two approaches were used, gland quarters to preserve cellular integrity (23) and cell cultures to study Ang II effects on individual cells. We demonstrated that stimulation of the AT2 receptor by Ang II or CGP42112 not only increased DNA fragmentation, but also induced cleavage of PARP, one of the earliest enzymes to be impaired in programmed cell death. Indeed, in the apoptotic process, DNA fragmentation is the result of an activation of deoxyribonucleases by caspases, and PARP cleavage is the result of caspase-3 activation (37, 38). This suggests that stimulation via AT2 receptor leads to the activation of the caspase-3 family of enzymes, supporting previous studies indicating that the AT2 receptor induced apoptosis (16, 17, 18, 19, 20, 21, 22, 23). The role of the AT1 receptor was also suggested. For example, in ventricular myocytes, apoptosis was mediated by AT1 receptor, but not by the AT2 receptor (50, 51, 52). However, in our studies, coincubation of Ang II with the AT1 receptor antagonist enhanced the effect of Ang II, indicating that the small proportion of AT1 receptors present in the fetal adrenal glands had no effect on apoptosis or antagonized the effect mediated by the AT2 receptor. These observations confirm previous studies of the opposite functions mediated by the two receptors (9, 10, 13).
Our immunofluorescence studies conducted on single cells clearly indicate that the fetal cells are targets of the AT2 receptor-induced apoptosis. Ang II caused actin reorganization, followed by important morphological changes, characterized by membrane blebbing. Cytoskeleton organization has been identified as a key regulator of apoptosis. Filamentous actin is necessary for blebbing and formation of apop-totic bodies (53). Our results indicate that after AT2 receptor stimulation, filamentous actin is more abundant at the base of blebs, corroborating previous observations conducted on stimulus-induced apoptotic cells (54, 55). These modifications of the actin network from cytoplasmic distribution to exclusive membrane localization may be due to the actions of caspases. Indeed, recent work suggests that actin-binding proteins, such as fodrin, gelsolin (56, 57), myosin (the myosin light chain) (58), and/or actin itself (56, 59), may be subject to degradation by caspases or calpains, another protease recently identified for its implication in membrane blebbing and cytoskeletal rearrangements (60).
All of these data indicate that the high expression of AT2 receptors in the fetal zone may be correlated with the high level of apoptosis present in this area of the adrenal gland during development. The molecular mechanism of the signaling pathway for AT2-conducted apoptosis remains to be determined in the fetal adrenal gland. In a number of studies, it has been shown that the AT2 receptor inhibits MAPK activity (p42mapk and p44mapk, ERK1/ERK2), an action mediated by activation of the tyrosine phosphatase SHP-1 in N1E-115 neuroblastoma cells (61), serine-threonine phosphatase PP2A in rat brain neuronal cultures (62), or mitogen-activated protein kinase phosphatase MKP1 in PC12W and R3T3 cells (16). With regard to apoptosis, Horiuchi et al. have shown that the AT2-mediated activation of MKP1 leads to the dephosphorylation of the antiapoptotic Bcl-2 and up-regulation of proapoptotic Bax (63, 64). In endothelial cells, AT2 receptor stimulation of apoptosis was associated with the activation of caspase-3, and nitric oxide donors completely reversed Ang II-induced apoptosis (20). Another recent study implicated ceramide as a new effector of AT2 receptor action (65).
How can a single factor, such as the AT2 receptor, control initiation of apoptosis (Refs. 16, 18, 19 and this study) and initiation of cell differentiation, such as steroid secretion (12, 18) or neurite elongation (13, 14)? In fact, all of these processes involve common or similar intracellular pathways that ultimately converge on mitogen-activated protein kinase activity. Transient vs. sustained activation or inhibition will, in fact, induce, respectively, proliferation, differentiation, or cell death. In addition to specific interactions with other hormones or growth factors, we propose that environmental cues could together orient cell decision through one of the three described functions. An action on microtubules, which are known to play an important role in apoptosis signaling, could also be expected (66). In the present study, we show that in vivo, apoptotic cells are localized in a zone of the fetal adrenal gland in which AT2 is strongly expressed. Our results also indicate that specific stimulation of the AT2 receptor promotes apoptosis in cultured fetal adrenal cells. However, no DNA fragmentation was detected before 16 weeks of fetal life, even though AT2 receptors are present throughout the entire cortex during this period. This may be explained by the fact that not all factors involved in the apoptotic pathway are present or functional at this age, or that other specific functions are associated with AT2 receptor activation, such as steroidogenic activity, as described in other tissues (11, 12, 18, 67). Indeed, one of the remarkable properties of the fetal zone is to produce large amounts of the androgen dihydroepiandrosterone during the second trimester of gestation, and this differentiated function is associated with a decrease in cell proliferation, an action in which the AT2 receptor is also known to be involved (10, 68, 69). The other action associated with AT2 receptor activation (in addition to an inhibitory effect on proliferation and promotion of apoptosis) is a role in neuronal differentiation (13, 14). Because AT2 receptors are also present on chromaffin cells, such an action could also be expected.
This work is the first to demonstrate hormonal induction of apoptosis in the human fetal adrenal gland. We demonstrate that Ang II stimulation, via the AT2 receptor, in cultured human fetal adrenal cells induces a rapid and sustained reorganization of the actin network. This rapid effect (within 1 min) suggests that the cytoskeleton is not only a target of Ang II action, but that it may also participate in the cellular signaling of the AT2 receptor. This actin reorganization is durable, indicating that AT2 receptor activation may also induce a cascade of cellular events leading to an activation of caspases targeting actin microfilaments. Taken together, our results indicate that apoptosis is present in human fetal adrenal glands as early as 17 weeks gestation and increases during at least the following 4 weeks. More importantly, our results indicate that activation of the AT2 receptor of Ang II stimulates this programmed cell death, an action antagonized by the AT1 receptor. The high level of expression of the AT2 receptor together with its drastic disappearance after birth clearly suggest that this receptor is involved in adrenal gland development.
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Acknowledgments |
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Footnotes |
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Received June 16, 1999.
Revised August 6, 1999.
Accepted September 13, 1999.
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References |
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7ß1) in muscle function and
survival. Disrupted expression in merosin-deficient congenital muscular
dystrophy. J Clin Invest. 100:1870–1881.[Medline]
q/11-subunit with cytoskeleton in
adrenal glomerulosa cells: role in receptor-effector coupling. Endocrinology. 138:3299–3307.This article has been cited by other articles:
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  E. Q. Haxhija, H. Yang, A. U. Spencer, H. Koga, X. Sun, and D. H. Teitelbaum Modulation of mouse intestinal epithelial cell turnover in the absence of angiotensin converting enzyme Am J Physiol Gastrointest Liver Physiol, July 1, 2008; 295(1): G88 - G98. [Abstract] [Full Text] [PDF]
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 S. Roy, M. Rached, and N. Gallo-Payet Differential Regulation of the Human Adrenocorticotropin Receptor [Melanocortin-2 Receptor (MC2R)] by Human MC2R Accessory Protein Isoforms {alpha} and {beta} in Isogenic Human Embryonic Kidney 293 Cells Mol. Endocrinol., July 1, 2007; 21(7): 1656 - 1669. [Abstract] [Full Text] [PDF]
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 H. Ishimoto, D. G. Ginzinger, T. Matsumoto, Y. Hattori, M. Furuya, K. Minegishi, M. Tanaka, Y. Yoshimura, and R. B. Jaffe Differential Zonal Expression and Adrenocorticotropin Regulation of Secreted Protein Acidic and Rich in Cysteine (SPARC), a Matricellular Protein, in the Midgestation Human Fetal Adrenal Gland: Implications for Adrenal Development J. Clin. Endocrinol. Metab., August 1, 2006; 91(8): 3208 - 3214. [Abstract] [Full Text] [PDF]
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M.-C. Battista, M. Otis, M. Cote, A. Laforest, M. Peter, E. Lalli, and N. Gallo-Payet Extracellular Matrix and Hormones Modulate DAX-1 Localization in the Human Fetal Adrenal Gland J. Clin. Endocrinol. Metab., September 1, 2005; 90(9): 5426 - 5431. [Abstract] [Full Text] [PDF]
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 L. Gendron, J.-F. Oligny, M. D. Payet, and N. Gallo-Payet Cyclic AMP-independent Involvement of Rap1/B-Raf in the Angiotensin II AT2 Receptor Signaling Pathway in NG108-15 Cells J. Biol. Chem., January 31, 2003; 278(6): 3606 - 3614. [Abstract] [Full Text] [PDF]
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M. D. Payet, L. Bilodeau, L. Breault, A. Fournier, L. Yon, H. Vaudry, and N. Gallo-Payet PAC1 Receptor Activation by PACAP-38 Mediates Ca2+ Release from a cAMP-dependent Pool in Human Fetal Adrenal Gland Chromaffin Cells J. Biol. Chem., January 10, 2003; 278(3): 1663 - 1670. [Abstract] [Full Text] [PDF]
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E. Chamoux, A. Narcy, J.-G. Lehoux, and N. Gallo-Payet Fibronectin, Laminin, and Collagen IV as Modulators of Cell Behavior during Adrenal Gland Development in the Human Fetus J. Clin. Endocrinol. Metab., April 1, 2002; 87(4): 1819 - 1828. [Abstract] [Full Text] [PDF]
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 H. Sugino, R. Ozono, S. Kurisu, H. Matsuura, M. Ishida, T. Oshima, M. Kambe, Y. Teranishi, H. Masaki, and H. Matsubara Apoptosis Is Not Increased in Myocardium Overexpressing Type 2 Angiotensin II Receptor in Transgenic Mice Hypertension, June 1, 2001; 37(6): 1394 - 1398. [Abstract] [Full Text] [PDF]
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 B. Peters, S. Clausmeyer, P. Teubner, N. Obermüller, B. Kränzlin, N. Gretz, T. Inagami, and J. Peters Changes of AT2 Receptor Levels in the Rat Adrenal Cortex and Medulla Induced by Bilateral Nephrectomy and Its Modulation by Circulating ANG II J. Histochem. Cytochem., May 1, 2001; 49(5): 649 - 656. [Abstract] [Full Text]
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E. Chamoux, L. Bolduc, J.-G. Lehoux, and N. Gallo-Payet Identification of Extracellular Matrix Components and Their Integrin Receptors in the Human Fetal Adrenal Gland J. Clin. Endocrinol. Metab., May 1, 2001; 86(5): 2090 - 2098. [Abstract] [Full Text]
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 L. Breault, E. Chamoux, J.-G. LeHoux, and N. Gallo-Payet Localization of G Protein {{alpha}}-Subunits in the Human Fetal Adrenal Gland Endocrinology, December 1, 2000; 141(12): 4334 - 4341. [Abstract] [Full Text] [PDF]
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