ARTICLE |
Correspondence to: Evelyne VilaPorcile, Biologie de la Cellule Neuroendocrine, Collège de France, 11 Place Marcelin Berthelot, 75231 Paris Cedex 05, France.
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Summary |
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In addition to the circulating reninangiotensin system (RAS), a local system has been postulated in the anterior pituitary because immunodetection of its components in various mammalian species. However, different cell types appear to be involved in different species, and there is no general consensus on the subcellular localization of prorenin, renin and angiotensinogen. In this ultrastructural study, we investigated and quantified the presence of these components using double or triple immunogold labeling methods, in all the immunologically identified glandular cell types of the rat anterior pituitary. In contrast to previous reports, all these components were identified not only in lactotropes and gonadotropes but also in somatotropes, corticotropes, and thyrotropes. The highest levels were detected in lactotropes and gonadotropes, and renin gave the greatest signal. Angiotensinogen, prorenin, and renin were co-localized in the secretory granules of all rat pituitary glandular cell types. The simultaneous detection of the substrate (angiotensinogen) and both its specific cleavage enzyme and its proenzyme within the same granule suggests intragranular processing of this component. Moreover, the localization of these three constituents in the secretory granules also suggests that, in the rat anterior pituitary, they follow the regulated secretory pathway. (J Histochem Cytochem 46:301311, 1998)
Key Words: angiotensinogen, prorenin, renin, anterior pituitary cells, immunogold, electron microscopy, rat (Wistar)
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Introduction |
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In the circulating reninangiotensin system (RAS), angiotensin I (AI) and angiotensin II (AII) are generated from hepatic angiotensinogen (AGT), after a cascade of proteolytic cleavages involving a rate-limiting enzyme, renin. Renin is produced by the cleavage of prorenin, originating from the juxtaglomerular cells of the kidney. Local RAS systems also exist in nonrenal tissues, such as brain (reviewed in
A local RAS system has been revealed in the anterior pituitary by immunodetection of some of its components in various species (reviewed in
The rat pituitary has been investigated extensively, mainly by light microscopic immunocytochemistry. Renin, angiotensin-converting enzyme (ACE), and AII appeared to be co-localized with ß-luteinizing hormone (ß-LH) in gonadotropes (
By contrast, in the human pituitary, RAS components were detected only in lactotropes, both in normal and adenomatous tissue (
In the lamb pituitary, AGT, renin, ACE, and AII were all detected in lactotropes, and this system is used as a model for immunoelectron microscopic detection of these components (
Therefore, the exact cellular localization of the RAS components is still controversial. There have been few studies on their subcellular localization, although secretory granules in rat gonadotropes (
We focused our interest on the secretory granule compartment, the last site before secretion. We determined whether AGT, prorenin, and renin, were present in rat pituitary by immunoelectron microscopy. We studied lactotropes and gonadotropes to clarify the discrepancies reported in the literature, and also somatotropes, corticotropes, and thyrotropes, which had not previously been investigated at the ultrastructural level. The immunogold postembedding technique is a very efficient method for simultaneous detection of both the RAS components and specific hormones. Using this method, a quantitative analysis of each RAS element within identified cell categories was possible. In addition, triple immunogold labeling techniques enabled detection of co-localizations of different RAS elements, not only at the cell type or subtype level, particularly in the different subtypes of lactotropes, but more precisely within some heterogeneous secretory granule populations, such as the granulations of gonadotropes.
These techniques allowed, for the first time, detection of AGT, prorenin, and renin in all the glandular cells of the rat pituitary and demonstrated that the substrate AGT and its cleavage enzyme and proenzyme are co-localized in the same secretory granules.
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Materials and Methods |
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Materials
Antisera.
Sources and specificities of the antibodies used in this study are summarized in Table 1.
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Gold Conjugates. Protein A-conjugated colloidal gold (Biocell; Cardiff, UK) was applied after incubation with polyclonal antibodies, whatever the species in which they were raised. Goat anti-mouse IgGs conjugated with colloidal gold (GAM; Biocell) were used after incubation with monoclonal antibodies. In general, small gold particles (5 nm) were used for the detection of pituitary hormones (diluted 1:50 to 1:75), and larger ones (10 and 15 nm) for detection of the RAS components (diluted 1:25 to 1:50).
Methods
Pituitaries.
Pituitaries from normal adult male Wistar rats (200 g; Iffa-Credo, l'Arbresle, France) were fixed in 4% paraformaldehyde supplemented with 0.05% glutaraldehyde for 1 hr at 4C. The anterior lobes were minced and fixed for another hour in fresh fixative at 4C. The fragments were then embedded in LR White (LRW), as described by
Immunogold Reactions. Ultrathin sections from LRW-embedded blocks were mounted on gold grids. Postembedding dual or triple immunogold labeling techniques were then applied for simultaneous detection of different RAS components and/or pituitary hormones. The detection of RAS components (AGT, prorenin, and renin) required longer incubation, from 4 to 5 hr at room temperature (RT) or 15 to 20 hr at 4C, compared to 12 hr at RT for hormone detection.
Grids were treated on drops of different reagents, as previously described (
For double immunostaining, the procedure was repeated on the other side (Side B) of the grid (
Triple immunostaining was performed according to
The multiple immunostaining techniques were repeated with a different order of antibody application and with gold conjugates of different sizes in each case. The sequence of application did not appear to alter the results. However, simple labeling always gave a stronger immunoreaction than double immunostaining.
Concerning controls, all the antibodies have been previously characterized (see Table 1). Immunocytochemical controls involved omission of either one or several specific antibodies from the above procedures, and no staining could be observed on control grids.
Quantification. Quantification of secretory granule labeling was performed on printed micrographs (final magnification x 50,000), by counting the gold particles present on all the granule sections in each immunocytochemically identified pituitary cell type. Results were expressed as the mean number of particles per granule, and SEM evaluated with the one-way ANOVA program, including Fischer's and Scheffé's tests, p<0.01 or 0.05). Gold particles were counted on more than 4500 secretory granules, in 100 pituitary glandular cell sections. Other labeled structures (vesicles or cisternae) were excluded from this quantification.
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Results |
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Several RAS components were detected in the glandular cells of the male rat anterior pituitary using this postembedding immunogold procedure. However, the intensity of the immunoreactions varied greatly depending on the cell type (or subtype) and the component tested. The two cell types exhibiting the most prominent labeling for all the RAS components studied were lactotropes and gonadotropes. The other glandular cell types (somatotropes, corticotropes and thyrotropes) were not labeled as intensely (Figure 1). The gold particles were mainly distributed in storage sites, such as secretory granules, and also in some vesicles in all the pituitary cell types.
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Lactotropes
AGT (Figure 2, Figure 3, and Figure 5), prorenin, and renin (Figure 4, Figure 6, and Figure 7) were detected in the secretory granules of all lactotrope subtypes, either in cells containing large and polymorphic granules ("classical" lactotropes; Figure 2) or in the other subtypes harboring smaller, spherical granulations (Figure 3 and Figure 5), which were recognizable as lactotropes only after specific immunolabeling. Prorenin and renin were the major components (Figure 1A), largely displayed in the mature granules (Figure 4) and in immature granules within the Golgi area (Figure 6). Moreover, in the "classical" lactotropes, renin was present in vesicles (Figure 4), sometimes appearing as "buds" on the limiting membrane of some secretory granules (Figure 4 and Figure 7), as well as in crinophagic structures (Figure 8). Some secretory granules did not contain any RAS component (Figure 2 and Figure 3), whereas a few structures exhibited prorenin or renin labeling only (Figure 4). In many granules, triple labeling provided evidence of co-localizations of AGT and prorenin or renin with prolactin (Figure 4 and Figure 5).
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Gonadotropes
Most gonadotropes were identified morphologically by the presence of two populations of granules. In ultrathin sections from LRW-embedded pituitaries, large granulations had a reticulated matrix, whereas the smaller ones were less dense and sometimes appeared "empty" (Figure 9 Figure 10 Figure 11). The labeling of the two populations was also different for ß-LH and RAS components. The immunoreactions were always more intense in the small granulations (Figure 9 Figure 10 Figure 11), and these latter were taken into account only for quantification (Figure 1B). Strong immunolabeling for renin (Figure 1B and Figure 9 Figure 10 Figure 11) was observed in the small granules as opposed to a weak response for prorenin (Figure 9 and Figure 10) and a moderate reaction for AGT (Figure 11). In the large granulations, very few gold particles were observed for all the RAS components tested (Figure 9 and Figure 10). In many gonadotropes, a third structure was observed that showed positive labeling for renin, whereas prorenin was hardly detectable and AGT was not detected at all, ß-LH being present in many of them (Figure 9). These structures were of similar size to the large granulations but were denser. They were not clearly defined and were irregular in shape.
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Somatotropes
Somatotropes were poorly labeled for AGT (Figure 12) and prorenin (Figure 14), displaying weaker and more diffuse immunoreactions than other pituitary cell types. Renin was more abundant (Figure 1C) and was also present in some vesicles (Figure 13) and "buds" on the limiting membrane of some mature granules (Figure 15).
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Corticotropes and Thyrotropes
After LRW embedding, these cells revealed secretory granules with a clear content and often an "empty" aspect, but, unlike gonadotropes, their granule populations were homogeneous in size. Double immunostaining was still necessary for unequivocal identification of the two cell types and detection of RAS components. After an immunoreaction for AGT, the response of corticotropes (Figure 1D and Figure 17) appeared higher than that of thyrotropes (Figure 1E and Figure 19). Conversely, renin was significantly more abundant in thyrotropes (Figure 1E and Figure 20) than in corticotropes (Figure 1D and Figure 16).
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Discussion |
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This work reports the existence of three RAS components in the storage compartment of rat anterior pituitary cells. All glandular cell types were found to contain AGT, prorenin, and renin, to different extents. AGT is most abundant in lactotropes and, in both lactotropes and gonadotropes, it co-localizes with prorenin and a significant amount of renin in secretory granules. AGT and prorenin are also present in somatotropes, corticotropes, and thyrotropes. Renin is more abundant in somatotropes than in the latter two cell types. Quantitative data show that the distribution of prorenin and renin in the storage compartment is significantly different from one pituitary cell type to another. This is particularly evident for lactotropes and gonadotropes, which present particular profiles (Figure 1A and Figure 1B) corresponding to a specific recognition of the two components by their related antibodies, i.e., 657-2 for prorenin and 2D12 for renin.
As already mentioned in the Introduction, previous studies with light microscopic immunocytochemistry failed to detect AGT or renin in rat pituitary lactotropes. In contrast, AGT, prorenin, and renin have been found in the secretory granules of human and lamb lactotropes by immunoelectron microscopy. Our results are therefore the first demonstration of the co-localization of these RAS components in rat pituitary lactotropes, suggesting that this co-localization is not species-specific. The presence of all three RAS components in rat anterior pituitary lactotropes suggests that at least part of the RAS is functional in this cell type. Processing of prorenin to renin appears to occur not only within mature secretory granules, as previously suggested for human pituitary lactotropes, which also contain cathepsin B (
Complementary experiments were performed with immunoperoxidase on GH3B6 cells, a lactotrope rat tumor cell line. Golgi saccules were labeled for prorenin but not for renin, which appeared to be located in secretory granules. These detections confirmed the specificity of the immunoreactions, and provided further arguments in favor of intragranular processing of prorenin to renin and against uptake of these components from the extracellular medium (unpublished results). Trafficking of renin-containing vesicles appears to occur in rat lactotropes, either fusing with or budding from mature granules. Such trafficking could be related either to a phenomenon of granule maturation (
The presence of AGT in rat gonadotropes reported here is in agreement with the previous results of
In this article, we present novel data indicating that rat somatotropes contain both AGT and renin. The level of renin is relatively high, with a renin:prorenin ratio of almost 3 (Figure 1C). It occurs within the secretory granules, in buds on the limiting membranes, and in many vesicles, suggesting intracellular trafficking of renin, as discussed above in lactotropes. The level of AGT in somatotropes was low and may reflect rapid cleavage by the high intracellular level of renin. Previous immunocytochemical investigations failed to detect AGT (
In rat pituitary corticotropes and thyrotropes, our accurate technique also enabled detection of low levels of AGT, prorenin, and renin, which has not been previously reported. Again, the presence of AII in these pituitary cell types has not yet been studied.
These data suggest that AGT, prorenin, and renin are sorted towards the regulated secretory pathway and processed in the secretory granules. This contradicts previous reports that AGT is secreted only constitutively by transfected AtT-20 cells (
In summary, we have shown for the first time that AGT, prorenin, and renin are present in all glandular cell types of the rat anterior pituitary. In addition, we have demonstrated that both the substrate AGT and its cleavage enzyme, renin, are co-expressed in the same secretory granules together with a significant amount of the proenzyme prorenin. This was particularly clear in lactotropes and gonadotropes. Because RAS components are known to interfere with the secretory activity of several pituitary cell types, and particularly that of lactotropes, corticotropes and thyrotropes (
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Acknowledgments |
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We would like to thank the National Hormone and Pituitary Program, the National Institute of Diabetes and Digestive and Kidney Diseases, the National Institute of Child Health and Human Development and the US Department of Agriculture for the gift of anti-ß-LH, anti-GH and anti-ß-TSH antisera. We are also indebted for their generous gifts of antibodies to J. Sealey (657-2), D. Simon (2D12), F. Pinet (ORNI), R. Counis (anti-oLHß), and D. Grouselle (anti-PRL). We are also grateful to J.M. Gasc for his help in gathering most of these antibodies, and many other ones!
We would like to thank A. TixierVidal for valuable advice and C. Tougard for helpful discussions. We acknowledge the technical help of R. Picart and A. Barret and the secretarial assistance of A. Bayon. We also thank E. Etienne for his expert photographic work.
Received for publication March 17, 1997; accepted September 3, 1997.
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