Abstract
In addition to the circulating renin–angiotensin 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. Angio-tensinogen, 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.
Keywords
I
A local RAS system has been revealed in the anterior pituitary by immunodetection of some of its components in various species (reviewed in Deschepper 1991; Saavedra 1992; Ganong 1993,1994,1995; reviewed in Sernia et al. 1997). These studies showed that pituitary cell types containing components of the RAS differed in different mammalian species.
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 β-luteinizing hormone (β-LH) in gonadotropes (Ganong et al. 1989; Naruse et al. 1986,1991). Co-localization of A II and β-LH was confirmed by immunoelectron microscopy (Deschepper et al. 1986a). The immunolocalization of AGT is controversial. Deschepper and Ganong (1991) and Ganong et al. (1989) reported that AGT is located in a separate population of cells and does not co-localize with any known anterior pituitary hormone. By contrast, Sernia et al. (1992,1997) and Thomas and Sernia (1990) detected AGT in gonadotropes and also in a population of perisinusoidal cells. In the rat pituitary, RAS components, either AGT (Ganong et al. 1989; Thomas and Sernia 1990; Deschepper and Ganong 1991; Sernia et al. 1992) or renin (Naruse et al. 1981,1986), were not detected in lactotropes. The presence of AII in lactotropes has been reported by some authors (Steele et al. 1982). However, other authors have failed to detect it (Deschepper et al. 1985; Naruse et al. 1986). Renin has not been detected in rat somatotropes (Naruse et al. 1981; Saint-André et al. 1989), corticotropes, or thyrotropes (Naruse et al. 1981). RAS constituents were not found in folliculo-stellate cells, identified by their immunoreactivity to the S-100 protein (Ganong et al. 1989; Thomas and Sernia 1990; Deschepper and Ganong 1991; Sernia et al. 1992,1997).
By contrast, in the human pituitary, RAS components were detected only in lactotropes, both in normal and adenomatous tissue (Mukai et al. 1984; Saint-André et al. 1986). The presence of prorenin (Saint-André et al. 1989) and renin (Saint-André et al. 1989; Rousselet et al. 1990) was also revealed in these cells by immunoelectron microscopy.
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 (Kettani et al. 1991).
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 (Deschepper et al. 1986a) and in human (Saint-André et al. 1989; Rousselet et al. 1990) and lamb (Kettani et al. 1991) lactotropes have been reported to contain RAS constituents.
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 soma-totropes, 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 gona-dotropes.
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 proen-zyme are co-localized in the same secretory granules.
Materials and Methods
Materials
Antisera. Sources and specificities of the antibodies used in this study are summarized in Table 1.
Gold Conjugates. Protein A-conjugated colloidal gold (Bio-cell; Cardiff, UK) was applied after incubation with poly-clonal 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).
Characterization of antisera used in this study
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% glutaralde-hyde 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 Ozawa et al. (1994) after Newman (1987).
Immunogold Reactions. Ultrathin sections from LRW-embedded blocks were mounted on gold grids. Postembed-ding 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 1–2 hr at RT for hormone detection.
Grids were treated on drops of different reagents, as previously described (Ozawa et al. 1994; Vila–Porcile and Barret 1996), using specific antibodies diluted in Tris-maleate buffer supplemented with 1% BSA and related colloidal gold conjugates in the same buffer (1–2 hr). Grids were air-dried, stained for 8–10 sec with 2% uranyl acetate in 50% ethanol, and examined under a LEO 906 electron microscope (Oberkochen, Germany).
For double immunostaining, the procedure was repeated on the other side (Side B) of the grid (Bendayan 1982) when completely dry, using another specific antibody and a colloidal gold conjugate of a different size. Uranyl acetate staining was always performed on the side treated first (Side A).
Triple immunostaining was performed according to Ozawa et al. (1994). After application of the first specific antibody and of its related immunogold conjugate on Side A, a 1-hr incubation with unconjugated protein A (0.2 mg/ml) was intercalated to block any protein A binding sites on the sections. The grid was then incubated with the second specific antibody and an immunogold conjugate of a different size was applied. After complete drying, a procedure similar to single immunolabeling was performed on the other side (Side B) of the grid, using a third specific antiserum and an immunogold conjugate of yet a different size.
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.
ORNI, anti-angiotensinogen (AGT) antibody; 657–2, anti-prorenin antibody; 2D12, anti-renin antibody. Values are significant for the following. (
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.
Results
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.
Lactotropes
AGT (Figures 2, 3, and 5), prorenin, and renin (Figures 4, 6, and 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 (Figures 3 and 5), which were recognizable as lactotropes only after specific im-munolabeling. 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 (Figures 4 and 7), as well as in crinophagic structures (Figure 8). Some secretory granules did not contain any RAS component (Figures 2 and 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 (Figures 4 and 5).
Gonadotropes
Most gonadotropes were identified morphologically by the presence of two populations of granules. In ul-trathin sections from LRW-embedded pituitaries, large granulations had a reticulated matrix, whereas the smaller ones were less dense and sometimes appeared “empty” (Figures 9–11). The labeling of the two populations was also different for β-LH and RAS components. The immunoreactions were always more intense in the small granulations (Figures 9–11), and these latter were taken into account only for quantification (Figure 1B). Strong immunolabeling for renin (Figures 1B and 9–11) was observed in the small granules as opposed to a weak response for prorenin (Figures 9 and 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 (Figures 9 and 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, β-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.
Co-localization of AGT (15 nm) and PRL (5 nm) in a lactotrope containing small secretory granules.
Triple labeling for renin (R, 10 nm), PRL (5 nm), and prorenin (pR, 15 nm) in a large-granulated lactotrope. Short arrows indicate a vesicle and a bud containing renin; large arrow indicates a structure containing prorenin only.
Co-localization of renin (R, 10 nm), PRL (5 nm), and AGT (15 nm).
Double labeling for renin (R, 10 nm) and prorenin (pR, 5 nm) on immature granules in the Golgi area of a cell identified morphologically as a lactotrope.
Double labeling for renin (R, 10 nm) and prorenin (pR, 5 nm). A bud is seen on the limiting membrane of a granule containing renin only (thick arrow). Thin arrows indicate two renin-containing vesicles.
A crinophagic vacuole in a lactotrope. Double staining: renin (R, 10 nm) and PRL (5 nm). Renin remains present within a granule included in a lytic vacuole (arrowhead) and in several vesicular structures (arrows).
Gonadotropes: postembedding immunodetection of RAS components with colloidal gold particles of different sizes. The sequence of application of the reagents and the size of the gold particles used are indicated on each micrograph. Bars = 0.2 μm.
Triple staining: renin (R, 10 nm), β-LH (LH, 5 nm), and prorenin (pR, 15 nm). Large granulations (arrows) are mainly labeled for β-LH, whereas the small granules contain β-LH, renin, and prorenin. Dense structures (curved arrows) are labeled either for both β-LH and renin (Figure 9) or for renin only (Figure 10) (see text).
Triple staining: renin (R, 10 nm), β-LH (LH, 5 nm), and AGT (15 nm). AGT co-localizes with renin and β-LH in most small granules.
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).
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 immu-nostaining 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 (Figures 1D and 17) appeared higher than that of thyrotropes (Figures 1E and 19). Conversely, renin was significantly more abundant in thyrotropes (Figures 1E and 20) than in corti-cotropes (Figures 1D and 16).
Discussion
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 lac-totropes and gonadotropes, it co-localizes with prore-nin and a significant amount of renin in secretory granules. AGT and prorenin are also present in soma-totropes, 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 (Figures 1A and 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 lacto-tropes. 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 (Saint-André et al. 1989), but also in immature granules of the Golgi area, in which both the proenzyme and the enzyme are co-localized (see Figure 6).
Complementary experiments were performed with immunoperoxidase on GH3B6 cells, a lactotrope rat tumor cell line. Golgi saccules were labeled for prore-nin 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 (Grimes and Kelly 1992) or to the existence in rat lac-totropes of a “constitutive-like secretory pathway” (Arvan and Castle 1992) operating in addition to the regulated secretory route.
The presence of AGT in rat gonadotropes reported here is in agreement with the previous results of Sernia et al. (1992,1997) and Thomas and Sernia (1990). These authors demonstrated AGT secretion by immu-nocytochemically identified gonadotropes, using the reverse hemolytic plaque assay (Sernia et al. 1992). Moreover, because they did not observe uptake of [125I]-AGT by these cells, they concluded that gonado-tropes synthesize AGT. These results were questioned by Ganong in his review articles (1993,1994,1995), because he and his collaborators did not find AGT in the gonadotropes but rather in other unidentified cell types (Ganong et al. 1989; Deschepper and Ganong 1991). In contrast, using the same ORNI anti-AGT antibody as Ganong et al., we obtained low but convincing labeling of the small secretory granules of rat gonadotropes. However, the conditions of fixation and antibody application were different. Our material was only partially dehydrated (70%) and was embedded in LRW, an acrylic resin that polymerizes at rather a low temperature (50C) and may preserve antigens better. It has also been suggested for AGT localization in brain that contradictory results from different laboratories may be due to differences in the methods of tissue processing (Campbell et al. 1991). The most prominent RAS component revealed in secretory granules of rat gonadotropes was renin, which was expressed in the small granulations eightfold more than was prorenin (Figure 1B), thus suggesting its rapid cleavage into renin. Renin is also present in ill-defined structures, sometimes containing hormones but with ultrastructural features reminiscent of lysosomes, which have not been previously described in the literature. This would be consistent with the ly-sosomal nature of some granulations in rat gonado-tropes (Tougard et al. 1974), which has also been postulated for lactotropes (Saint-André et al. 1989) and for renin-secreting cells in the kidney (Taugner et al. 1985; Hackenthal et al. 1990; Reudelhuber et al. 1993). Many authors have reported that gonado-tropes also contain AII (Steele et al. 1982; Deschepper et al. 1986a; Naruse et al. 1986; Ganong et al. 1989; Thomas and Sernia 1990; Sernia et al. 1997), and it is possible that AGT is processed to AII in these cells. The detection of renin mRNA by in situ hybridization experiments in cells identified as gonadotropes (De-schepper et al. 1986b) provides further evidence against an internalization of circulating RAS components in pituitary cells. Furthermore, AGT mRNA has also been detected in pituitary cell extracts, both by a solution hybridization assay (Hellmann et al. 1988) and by RT-PCR (Sernia 1995).
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 (Ganong et al. 1989; Deschepper and Ganong 1991) and renin in rat (Naruse et al. 1981) and human (Saint-André et al. 1989) somato-tropes. The presence of AII in somatotropes has not yet been investigated.
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 con-stitutively by transfected AtT-20 cells (Deschepper and Reudelhuber 1990) and by in vitro preparations from bovine pituitaries (Colomer et al. 1996). Trans-fection experiments with the human prorenin gene in AtT-20 cells also suggested constitutive expression of prorenin (Pratt et al. 1988). Conversely, our results are in agreement with data from transfection experiments with the human renin gene (Fritz et al. 1987; Pratt et al. 1988) and the mouse Ren-2 gene (Laden-heim et al. 1989) in AtT-20 cells. Two pathways were observed in these transfected cells. Some prorenin was constitutively secreted (Pratt et al. 1988) but another portion was also sorted into secretory granules, together with renin (Fritz et al. 1987; Ladenheim et al. 1989), and this sorting depended on an interaction with a subclass of proteases (Brechler et al. 1996). These experiments suggest a “constitutive-like” pathway for renin, which is further supported by the presence of renin-containing vesicles in different pituitary cell types and would be in addition to the regulated secretory route.
Double staining for renin (R, 10 nm) and GH (5 nm). Renin was detected in most of the secretory granules together with GH, but occured alone in some vesicular structures (arrows).
Double staining for prorenin (pR, 10 nm) and GH (5 nm). Only a few 10-nm gold particles are present on the secretory granules immunostained for GH (arrows).
Labeling for renin (R, 10 nm) in a somatotrope, identified morphologically, showing a renin-containing bud on the limiting membrane of a mature secretory granule (arrow).
Double staining: AGT (10 nm; arrows) and ACTH (5 nm).
Double staining: renin (R, 10 nm; arrow) and ACTH (5 nm).
Double staining: renin (R, 10 nm; arrows) and β-TSH (5 nm).
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 thy-rotropes (Saavedra 1992; reviewed in Ganong 1995), further work is needed to determine whether these cells synthesize and secrete angiotensins, to establish whether the entire functional system is present within the glandular cells of the rat anterior pituitary.
Footnotes
Acknowledgements
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-β-LH, anti-GH and anti-β-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-oLHβ), 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. Tixier-Vidal 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.