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Endocrinology Vol. 141, No. 12 4334-4341
Copyright © 2000 by The Endocrine Society

ARTICLES

Localization of G Protein {alpha}-Subunits in the Human Fetal Adrenal Gland1

Lyne Breault2, Estelle Chamoux, Jean-Guy LeHoux3 and Nicole Gallo-Payet

Service of Endocrinology (L.B., E.C., N.G.-P.), Department of Biochemistry (J.-G.L), Faculty of Medicine, University of Sherbrooke, Sherbrooke, Québec, Canada J1H 5N4

Address all correspondence and requests for reprints to: Dr. Nicole Gallo-Payet, Service of Endocrinology, Faculty of Medicine, Université de Sherbrooke, 3001, 12th Avenue North, Sherbrooke, Québec, Canada J1H 5N4. E-mail: n.gallo{at}courrier.usherb.ca


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
The aim of the present study was to investigate the presence and localization of the main G protein {alpha}-subunits in the human fetal adrenal gland during the second trimester of gestation. Immunofluorescence studies conducted on sections from frozen glands obtained immediately after therapeutic abortion indicated that the {alpha}s subunit of the heterotrimeric Gs protein was detected in all adrenal cell types, except for endothelial cells. The other {alpha}-subunits had a more specific pattern of distribution. Indeed, the {alpha}i1–2 protein was restricted to the definitive zone, whereas {alpha}i3 labeling was mainly expressed in the fetal zone. The {alpha}q protein subunit was localized in vascular endothelial cells at the periphery of the adrenal gland and in fetal cells at the center. Finally, chromaffin cells expressed {alpha}s, {alpha}q, and {alpha}o1, but not {alpha}o2 nor {alpha}i. Altogether, these results indicate that the human fetal adrenal gland is not only unique in its particular morphology and expression of steroidogenic enzymes, but also by the differential expression of G protein {alpha}-subunits. Such cell specific distribution in glands from midgestational fetuses may account for the absence or the different responses to stimuli, when compared with the adult adrenal gland.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
MOST SEVEN membrane-spanning receptors activate second messenger production by coupling to heterotrimeric G proteins. These particular proteins are composed of {alpha}- and ß{gamma}-subunits. Ligand binding to the receptor catalyzes the exchange of GDP for GTP on the {alpha}-subunit and the dissociation into {alpha}-GTP and ß-{gamma}-subunits. Receptor interaction with {alpha}-subunits generates the classical second messengers cAMP, inositol phosphates and diacylglycerol (1, 2, 3). G{alpha} subunits belong to four main classes: those which stimulate (G{alpha}s family) or inhibit (G{alpha}i/G{alpha}o family) adenylyl cyclase activity, those which stimulate phospholipase C activation (G{alpha}q family) and those mediating calcium and potassium channel activities (G{alpha}o1 and G{alpha}o2).

Two of the main stimuli of the adult adrenal gland are ACTH and angiotensin II (Ang II), for which receptors belong to the class of G protein-coupled-receptors (GPCR). However, their respective roles during fetal adrenal development are not completely characterized. Moreover, both morphology and function of the human adrenal gland have specific properties which are not found in rodents. The primate fetal adrenal cortex is composed primarily of two zones: the outer definitive zone or neocortex and the larger inner fetal zone, comprising over 85% of total adrenal volume (4, 5). Enlargement of the fetal zone mainly accounts for the rapid growth of this gland during pregnancy. Cells from the fetal zone form radial cords and increase in size from the periphery to the central region of the gland where they are loosely distributed between sinusoids. These cells show ultrastructural features characteristic of steroid-secreting cells, producing large amounts of dehydroepiandrosterone sulfate (DHEAS). Cells from the definitive zone are arranged in small clusters separated by capillaries. These cells have scanty cytoplasm and are proliferative rather than steroidogenic. In addition to proliferation and differentiation, development of the human fetal adrenal gland involves programmed cell death occurring primarily in the central region of the gland (6, 7). A third zone, named the transitional zone, differentiates between the definitive zone and fetal zone at the end of the second trimester. During late gestation, the fetal cortex shows adult-like functional characteristics. Cells from the definitive zone express the enzymes required for aldosterone synthesis, whereas the transitional zone may produce cortisol with fetal cells still secreting DHEAS (reviewed in Ref. 8). After birth, the adrenal gland undergoes significant remodeling. The volume of the fetal zone decreases and the cortex gradually assumes the morphological organization of the adult cortex.

While cortical cells stem from the mesoderm, chromaffin cells forming the adrenal medulla have an ectodermal origin. During fetal development, pheochromoblasts originating from the neural crest migrate inside the fetal cortex to invade the central portion of the gland along with the developing sinusoids and the centro-medullary vein (9). During their migration through the fetal cortex, paracrine action of steroids induces their differentiation in endocrine chromaffin cells (10).

ACTH is one of the key players regulating growth and steroidogenesis of the fetal adrenal gland (11, 12, 13, 14), at least in the fetal zone. However, observations of human anencephalic fetuses and studies conducted on baboons (15, 16) have led to the concept that development of the definitive zone is relatively independent from ACTH. In addition, other factors produced systemically or locally, have since recently been identified, such as CRH (17), growth factors (18, 19), and angiotensin II. Indeed, we have shown that both Ang II type 1 (AT1) and type 2 (AT2) receptors are present in the human fetal adrenal gland between 16 and 20 weeks of gestation (20). While AT2 receptors in the fetal zone are associated with apoptosis (7), there are conflicting data regarding the effects of the AT1 receptors located in the definitive zone (14, 20, 21, 22). In none of these studies has the nature or localization of the coupling proteins been identified. Specificity, amplitude and rapidity of cellular responses generated by GPCRs depend on signal transduction mediated by these G{alpha} subunits. Thus, the presence or absence of specific G{alpha} subunits on a particular cell type will modulate its responsiveness to a given stimulus.

The aim of the present study was therefore to investigate the localization of {alpha} subunits linked to classical second messengers, namely {alpha}s, {alpha}i, {alpha}q, and {alpha}o in glands obtained immediately following therapeutic abortion. The results indicate that the human fetal adrenal gland is not only unique in its particular morphology and expression of steroidogenic enzymes, but also by the differential expression of the G protein {alpha}-subunits. These observations contribute to our understanding of the coupling process of factors involved in the development of the human fetal adrenal gland during midgestation.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Chemicals
The chemicals used in the present study were obtained from the following sources: antirabbit Ig-fluorescein from Amersham Pharmacia Biotech (Oakville, Ontario, Canada); enhanced chemiluminescence (ECL) detection system from Roche Molecular Biochemicals (Montréal, Québec, Canada); BioMax MR single emulsion films from Kodak (Rochester, NY); Bio-Rad Laboratories, Inc. DC protein assay kit from Bio-Rad Laboratories, Inc. (Mississauga, Ontario, Canada); polyvinylidene difluoride (PVDF) membranes from Millipore Corp. (Bedford, CA); antirabbit IgG coupled to horse radish peroxydase and Vectashield mounting medium from Vector Laboratories, Inc. (Burlingame, CA). Primary antibodies directed against G{alpha}s were purchased from NEN Life Science Products (Boston, MA) and antibodies against G{alpha}q were kindly provided by Drs. Marie-Noëlle Dufour and Gilles Guillon (CNRS-INSERM U469, Montpellier, France). These antibodies have no cross-reactivity with any of the other members of the G{alpha} family. G{alpha}i antibodies were purchased from Calbiochem (San Diego, CA). G{alpha}i1 antibody binds only to the {alpha}i1 isoform, whereas G{alpha}i1–2 recognizes both {alpha}i1 and {alpha}i2 isoforms. According to the Calbiochem company, G{alpha}i3 antibody has no cross-reactivity with any of the other members of the G{alpha} family. Antibodies raised against G{alpha}o1 and G{alpha}o2 were a kind gift from by Dr. Vincent Homburger (CNRS-INSERM U469, Montpellier, France) and have no cross-reactivity against each other nor against the other G{alpha} family members. Antibody directed against the von Willerbrand factor (vWF) was purchased from DAKO Corp. (Mississauga, Ontario, Canada).

Retrieval and preparation of glands
Fetal adrenal glands were obtained from fetuses aged 16–20 weeks (post fertilization) at the time of therapeutic abortion. Fetal ages were estimated by foot length and time after last menstruation, according to Streeter et al. (23). 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 or fixed in 4% paraformaldehyde.

Western blotting
Whole fetal glands and adult rat brains were homogenized in boiling phosphate buffer, pH 7.6, containing 1% SDS for total protein preparation. Proteins were quantified using the Bio-Rad Laboratories, Inc. DC protein assay kit and extracts were stored at -20 C for subsequent utilization. For Western blot electrophoresis, proteins were boiled for 5 min in 1% SDS, 5% ß-mercaptoethanol and separated (10 µg per lane and 20 µg for detection of {alpha}o) by SDS-PAGE [10% (wt/vol) acrylamide]. Proteins on gel were electrotransferred onto PVDF membranes in Tris-glycine buffer containing 20% methanol. Membranes were then washed in Tris buffered saline (TBS)-Tween 20 (0.05%) and blocked with 1% gelatin in TBS-Tween 20 for 2 h at room temperature. Primary antibodies directed against G protein {alpha}-subunits were diluted (1:2000) in 0.1% BSA/TBS-Tween 20 and blots were incubated overnight at 4 C. Blots were finally incubated 1 h at room temperature with antirabbit IgG coupled to horse radish peroxydase to visualize immunopositive bands on BioMax MR film using the ECL Western blotting system.

Immunolocalization
Whole glands were immersed immediately after removal in 4% paraformaldehyde for 24 h, embedded in paraffin and cut into 5-µm sections. Sections were deparaffinized using standard histological procedures, heat-treated in 0.01 M citric acid and incubated in 0.1 M glycine for 30 min at 4 C. Non specific binding was blocked by 30 min incubation in phosphate buffer containing 5% nonfat milk, at 4 C. Primary antibodies against G{alpha} subunits and von Willerbrand Factor were diluted (1:50) in HBS (NaCl, 130 mM; KCl, 3.5 mM; CaCl2, 1.8 mM; MgCl2, 0.5 mM; NaHCO3, 2.5 mM; HEPES, 5 mM) 5% nonfat milk and incubated for 60 min at room temperature. The sections were then incubated with antirabbit IgG-FITC for 60 min at room temperature and mounted with Vectashield medium. Fluorescent labeling was visualized on a Nikon DM 400 microscope equipped for epifluorescence using B-1E FITC filter set (Nikon, Melville, NY).


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Localization of {alpha}-subunits of heterotrimeric G proteins in midgestational human fetal adrenal glands
We examined the distribution of the different {alpha} subunits in glands from 16–20 weeks of gestation, obtained immediately after therapeutic abortion. The protein subunit {alpha}s was expressed in both the definitive zone (Fig. 1Go, A and B) and fetal zone (Fig. 1AGo, arrow and C). However, labeling was different between the two cell types. The small definitive cells, which have a high nucleus/cytoplasm ratio compared with the fetal cells, expressed {alpha}s uniformly, but less intensely (Fig. 1BGo) than the large fetal cells (Fig. 1Go, A and C), where maximal intensity approached the central portion of the gland (Fig. 1Go, C and D). In addition, the transitional zone between definitive and fetal zones had an attenuated level of labeling (Fig. 1AGo). As attested by corresponding chromogranin A labeling (Fig. 1EGo), the islets of chromaffin cells dispersed in the fetal zone were also immunoreactive for {alpha}s (Fig. 1DGo).



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  		Figure 1. Immunofluorescent labeling of the {alpha}s protein in the human fetal adrenal gland (17 weeks). Whole gland sections were fixed in paraformaldehyde 4% for 20 min and processed for immunofluorescent localization using {alpha}s antibody and antirabbit IgG-FITC as described in Materials and Methods. A, The external portion of the gland, showing definitive (DZ) and fetal zone (FZ); B, higher magnification of the definitive zone; C, fetal cells, near the central portion of the gland; D–E: islets of chromaffin cells (CC) in the center of the gland, showing {alpha}s (D) or chromogranin A labeling (E). The upper right panel is a schematic adrenal gland section indicating the location of the different photomicrographs. Images are representative illustrations of three different experiments using human fetal adrenal glands aged between 16–20 weeks of gestation. Scale bars, (A,10 µm; B, C, 20 µm; D, E, 15 µm).

 
The immunolocalization of {alpha}q was completely different, being associated with capillaries and sinusoids rather than steroidogenic cells, at least in the definitive zone (Fig. 2AGo) and in the external portion of the fetal zone (Fig. 2BGo). Confirming this observation, {alpha}q labeling had a similar pattern of localization to that of the von Willerbrand factor (a marker of endothelial cells) (Fig. 2CGo). Approaching the center of the gland, the periphery of each fetal (Fig. 2DGo) and chromaffin cells (Fig. 2EGo) was also labeled.



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  		Figure 2. Immunofluorescent labeling of {alpha}q protein and von Willerbrand Factor (vWF) in the human fetal adrenal gland (19 weeks). Paraffin-embedded sections were used for localization of {alpha}q and vWF factor as described in Materials and Methods and legend of Fig. 1Go. A, The external portion of the gland, showing {alpha}q labeling associated with capillaries and sinusoids in the definitive (DZ) and fetal zone (FZ); B, higher magnification of fetal cells in the external part of the gland; C, immunofluorescent detection of vWF factor; D, {alpha}q labeling in fetal cells located in the central portion of the gland; E, labeling of {alpha}q in islets of chromaffin cells (CC) and in surrounding fetal cells. The lower right panel is a schematic adrenal gland section indicating the location of the different photomicrographs. Images are representative illustrations of three different experiments using human fetal adrenal glands aged between 16–20 weeks of gestation. Scale bars, (A, C, E, 10 µm; B, D, 30 µm).

 
Labeling of {alpha}i1–2 protein was observed in the majority of definitive cells, and, to a lesser extent, in the fetal zone (Fig. 3Go, A and B). In contrast, the expression of {alpha}i3 isoform was intense in the fetal zone (Fig. 3Go, C and D) but weak in the definitive zone (Fig. 3CGo). Both isoforms were absent in chromaffin cells (Fig. 3EGo). Moreover, {alpha}i3 labeling appeared associated with cytoplasmic structures (Fig. 3DGo). As these structures were abundantly expressed in the fetal zone, the latter appeared strongly labeled compared with the definitive zone.



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  		Figure 3. Immunofluorescent labeling of {alpha}i1–2 (A–B) and {alpha}i3 (C–E) in the human fetal adrenal gland (17 weeks). Paraffin-embedded sections were processed for immunofluorescent localization using {alpha}i1–2 or {alpha}i3 antibodies and antirabbit IgG-FITC as described in Materials and Methods. A, The external portion of the gland, showing definitive (DZ) and fetal zone (FZ); B, higher magnification of {alpha}i1–2 labeling in the definitive zone; C, {alpha}i3 labeling in the definitive zone; D, {alpha}i3 labeling in the fetal zone; E, absence of {alpha}i3 labeling in the islets of chromaffin cells (CC). The lower right panel is a schematic adrenal gland section indicating the location of the different photomicrographs. Images are representative illustrations of three different experiments using human fetal adrenal glands aged between 16–20 weeks of gestation. Scale bars, (A, C, E, 10 µm; B, 15 µm; D, 30 µm).

 
The neuronal {alpha}o1 subunit (Fig. 4AGo), but not {alpha}o2 (Fig. 4BGo), was expressed in the islets of chromaffin cells and in endothelial cells (arrowhead, Fig. 4AGo).



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  		Figure 4. Immunofluorescence labeling of {alpha}o1 (A) and {alpha}o2 (B) in the human fetal adrenal gland (17 weeks). Whole gland sections were fixed in paraformaldehyde 4% for 20 min and processed for immunofluorescent localization using {alpha}o1 or {alpha}o2 antibodies and antirabbit IgG-FITC as described in Materials and Methods. A, {alpha}o1 labeling in the islets of chromaffin cells (CC) and endothelial cells (arrow); B, absence of {alpha}o2 labeling in chromaffin cells (CC) and in fetal cells (FZ); CMV, central medullary vein. Images are representative illustrations of three different experiments using human fetal adrenal glands aged between 16–20 weeks of gestation. Scale bars, (A, B, 10 µm).

 
Western blotting analyses of G{alpha} subunits in midgestational human fetal adrenal glands
We also assessed the presence of G{alpha} subunits in total protein extracts from 16 to 20 week-old human fetal adrenal glands (Fig. 5Go). Antibody against {alpha}s protein revealed the presence of two specific bands of 45 and 52 kDa in the fetal adrenal as well as in the adult adrenal gland and adult rat brain. The anti-{alpha}q antibody detected a single band with an apparent molecular mass of 42 kDa in the fetal adrenal gland whereas two bands, corresponding to {alpha}q (lower) and {alpha}11 (upper) isoforms, were detected in the adult adrenal gland. Levels of {alpha}q in the fetal adrenal gland increased from 16–20 weeks of gestation. The proteins {alpha}o1 and {alpha}o2 were detected as single bands with an apparent molecular mass of 39 kDa. {alpha}o1 was present in extracts from fetal and adult adrenal glands and rat brain, corroborating the observations from immunofluorescence data. Levels of {alpha}o1 in the fetal gland decreased from 16–20 weeks of gestation but were still higher than in the adult adrenal gland. In contrast, {alpha}o2 was not detected in the fetal adrenal gland between 16 and 20 weeks but was present in the adult gland and rat brain. The {alpha}i1–2 antibody revealed the presence of 2 bands [the upper corresponding to {alpha}i1 (weak signal) and the lower to {alpha}i2 (approximately 40 kDa)] in the fetal adrenal gland, demonstrating the predominance of {alpha}i2 over {alpha}i1. The {alpha}i3 antibody revealed the presence of three bands, the major band at 41 kDa corresponding probably to {alpha}i3.



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  		Figure 5. Western blot analyses of G protein {alpha} subunits in midgestational human fetal adrenal glands. Total proteins (10 µg/lane except for {alpha}o1 and {alpha}o2, 20 µg/lane) were separated by electrophoresis and blotted with antibodies directed against the different {alpha} subunits, as indicated on the left. G proteins were detected by chemiluminescence as described in Materials and Methods. Human adult adrenal gland (HA) and whole rat brain (RB) proteins were used as controls. 16–20 w: proteins from 16- to 20-week-old human fetal adrenal glands. Numbers on the right indicate molecular mass (kilodaltons) assessed by the use of markers. Data are representative of five other experiments using human fetal adrenal glands aged between 16–20 weeks of gestation.

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
Our results are the first to document the localization of the main {alpha}-subunits of the heterotrimeric G proteins in fetal adrenal glands from midgestational human fetuses. Using glands processed immediately after removal, we demonstrated that, aside from {alpha}s, which is present in all adrenal cell types, the other {alpha}-subunits had a more specific cell distribution. Such observations may account for the varying responses to stimuli observed between adult and fetal adrenal glands.

Immunofluorescent localization of {alpha}s in definitive and fetal zones is consistent with the concept that cAMP, the second messenger generated from G{alpha}s-coupled receptor, is an important mediator of adrenal gland development, mainly through ACTH stimulation (8, 11). Indeed, we have found (data not shown) that ACTH strongly stimulates cAMP accumulation and steroid secretion in cultured cells, as reported by others (13, 14, 24). Immunofluorescence labeling of {alpha}s is clearly localized at the periphery of fetal cells, whereas labeling is more diffuse around the definitive cells. These two types of immunoreactivity could correspond to the presence of the two isoforms, {alpha}s-L (52 kDa) and {alpha}s-s (45 kDa) (25), which may have more specific cellular distribution. In this regard, we have previously shown that, in adult glomerulosa cells, {alpha}s is present not only at the membrane level, but also throughout the cytoplasm (26). In addition, the lower expression of {alpha}s in the definitive zone supports several observations, indicating that the proliferative activity of the definitive zone is independent from ACTH stimulation (5, 15, 16), but rather dependent of growth factors, acting through tyrosine kinase receptors, hence independently of G protein coupling (8, 12).

Interestingly, the {alpha}q protein, but not {alpha}s, is obviously associated with capillaries and sinusoids, at least in the external portion of the gland. The presence of {alpha}q on vessels suggests that control of vascular functions by neurotransmitters or neuropeptides from innervating fibers (27) is operative early during gestation and may control adrenal gland development. In the adult gland, appropriate neural stimulation induces endothelial cells to produce several peptides which can, in turn, act as paracrine factors on cortical cells. Among these are adrenomedullin, endothelin, and Ang II (28, 29, 30). Surprisingly, cell expression of the {alpha}q protein changes from the periphery to the central portion of the gland, where it becomes clearly associated with the fetal cells. These locations are thus compatible with the observations that CRH could directly stimulate fetal cells to produce DHEA/S through inositol phosphate production and protein kinase C activation (17, 31), a stimulation which could occur via {alpha}q or {alpha}i proteins. Indeed, {alpha}q protein is not detected in the definitive cells, although AT1 receptors of Ang II, a well-known Gq-coupled receptor, are present during the second trimester of gestation (20, 22). Such differential expression between hormone receptor and its coupling protein could explain conflicting data concerning the steroidogenic capacity of Ang II in the fetal adrenal gland. Experimental conditions surrounding isolated vs. cultured cells are another source of discrepancies, the latter favoring expression of AT1 receptors (20) and of {alpha}q (data not shown) (21, 32, 33, 34). In agreement with this hypothesis, Western blot analyses indicate the presence of only a single band corresponding to {alpha}q, whereas the two isoforms, {alpha}q and {alpha}11 are detected in the adult adrenal gland, corroborating the fact that ontogenesis of Gq proteins is not achieved during the second trimester of gestation. Alternatively, whether activation of AT1 receptors stimulates InsPs production, this could occur via {alpha}i2 protein, highly expressed in the definitive zone. AT1 receptor coupled to Gi2 protein may also participate in the growth-promoting activity of the definitive zone, through activation of the MAP kinase cascade, as demonstrated in other cell types (for review see Refs. 35, 36).

On the contrary, the {alpha}i3 Gi protein exhibits strong labeling in the fetal zone. We have previously shown that this region also contains a large number of Ang II receptors of the AT2 type (20) and that activation of these receptors induces apoptosis of the fetal cells (7), as described in the neuronal cell line PC12W (37) and in neuronal cultures (38). From these observations, it can be hypothesized that, in the fetal adrenal gland, the action of AT2 receptors could be mediated by {alpha}i3 protein, as demonstrated in some studies (39, 40). Immunofluorescent labeling of {alpha}i3 protein appears mainly associated with intracellular organelles. This particular localization may be related to a role for {alpha}i3 subunits in intracellular membrane trafficking observed by others (41, 42).

The isoform {alpha}o proteins are expressed by cells having neuroectodermal origins and, as expected, immunofluorescent labeling of {alpha}o1 is present in chromaffin cells. Among the two known isoforms, only the {alpha}o1 isoform is detected in the human fetal adrenal gland. In contrast, both {alpha}o1 and {alpha}o2 are detected in the adult gland. An age-related expression of {alpha}o isoforms has also been described in the rat brain (43). However, in this latter study, {alpha}o2 was found to be expressed early in development and {alpha}o1 during adult life. These discrepancies in {alpha}o isoform expression might reflect tissue or species differences. This suggests a particular function for {alpha}o1 during development of the human adrenal medulla, which may be related to cell migration, as described for embryonic neurons (44). The strong {alpha}o1 labeling of chromaffin cells represents a new and excellent morphological tool allowing for the localization of these cells during their migration through the developing fetal adrenal gland. Chromaffin cells also express {alpha}s and {alpha}q, suggesting that the physiology of these cells may be controlled by a variety of factors using these coupling proteins; among which pituitary adenylate cyclase-activating polypeptide (PACAP) is a strong candidate (45). However, {alpha}i proteins are not detected in chromaffin cells. Of note, whereas the AT2 receptor is present in these cells, no apoptotic figures are seen (unpublished observations). These results reinforce the idea that the presence of both receptor and G protein are necessary for functional coupling to second messenger production.

In summary, the results of this study are the first to document the presence and localization of the main {alpha} subunits of the heterotrimeric G proteins in the human fetal adrenal gland. Apart from the presence of {alpha}s in all cell types, we show that {alpha}i1–2 labeling is restricted to neocortical cells, whereas {alpha}i3 is mainly expressed in the fetal zone, and the {alpha}q subunit, but not {alpha}s, is associated with vascularization. All these results illustrating cell specific expressions of {alpha}-subunits could provide explanation for the different responses to stimuli (such as Ang II) observed between the adult and fetal adrenal gland.


   Acknowledgments
 
The authors would like to thank Ms. Lucie Chouinard for her skillful technical assistance, Dr. Vincent Homburger (CNRS-INSERM U469, Montpellier, France) for the gift of G{alpha}o1 and G{alpha}o2 antibodies, Drs. Marie-Noëlle Dufour and Gilles Guillon (CNRS-INSERM U469, Montpellier, France) for the gift of G{alpha}q antibody, and Dr. Daniel Ménard and Ms. Lina Corriveau (Department of Cell Biology, University of Sherbrooke) for providing fetal adrenal glands.


   Footnotes
 
1 This work was supported by Medical Research Council of Canada grants (to N.G.P. and J.G.L.) and the Fonds pour les Chercheurs et Aide à la Recherche. Back

2 Recipient of a Medical Research Council studentship. Back

3 A "chercheur boursier de carrière" of the FRSQ (Fonds pour la Recherche en Santé du Québec). Back

Received July 14, 2000.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

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