ARTICLE

Angiotensinogen, Prorenin, and Renin Are Co-localized in the Secretory Granules of All Glandular Cells of the Rat Anterior Pituitary: An Immunoultrastructural Study

Correspondence to: Evelyne Vila–Porcile, Biologie de la Cellule Neuroendocrine, Collège de France, 11 Place Marcelin Berthelot, 75231 Paris Cedex 05, France.

	Summary

Top Summary Introduction Materials and Methods Results Discussion Literature Cited

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. 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 46:301–311, 1998)

Key Words: angiotensinogen, prorenin, renin, anterior pituitary cells, immunogold, electron microscopy, rat (Wistar)

	Introduction

Top Summary Introduction Materials and Methods Results Discussion Literature Cited

In the circulating renin–angiotensin 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 Ganong 1993 ), heart, gonads, and various endocrine and exocrine glands (Deschepper and Ganong 1988 ; reviewed in Phillips et al. 1993 ).

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 , Ganong 1994 , Ganong 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 ß-luteinizing hormone (ß-LH) in gonadotropes (Ganong et al. 1989 ; Naruse et al. 1986 ,1991). Co-localization of A II and ß-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 , Sernia et al. 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 , Naruse et al. 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-Andre et al. 1989 ), corticotropes, or thyrotropes (Naruse et al. 1981 ). RAS constituents were not found in folliculostellate 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 , Sernia et al. 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-Andre et al. 1986 ). The presence of prorenin (Saint-Andre et al. 1989 ) and renin (Saint-Andre 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-Andre 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 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.

	Materials and Methods

Top Summary Introduction Materials and Methods Results Discussion Literature Cited

Materials
Antisera. Sources and specificities of the antibodies used in this study are summarized in Table 1.

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 Ozawa et al. 1994 after Newman 1987 .

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 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.

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

Top Summary Introduction Materials and Methods Results Discussion Literature Cited

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.

View larger version (14K): [in this window] [in a new window]   Figure 1. ORNI, anti-angiotensinogen (AGT) antibody; 657-2, anti-prorenin antibody; 2D12, anti-renin antibody. Values are significant for the following. (A) ORNI vs 657-2 (p<0.05) and vs 2D12 (p<0.01); 657-2 vs 2D12 (p<0.01). (B) ORNI and 657-2 vs 2D12 (p<0.01). (C) ORNI and 657-2 vs 2D12 (p<0.01). (D) ORNI vs 657-2 and 2D12 (p<0.01). (E) ORNI and 657-2 vs 2D12 (p<0.05).

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).

View larger version (144K): [in this window] [in a new window]   Figures 2-8. Lactotropes: 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. Figure 2. Double immunostaining for angiotensinogen (AGT, 15 nm) and prolactin (PRL, 5 nm), in a lactotrope with large secretory granules. Granules indicated by arrows are enlarged (x 80,000) in the inset to show more clearly the 5-nm gold particles. Figure 3. Co-localization of AGT (15 nm) and PRL (5 nm) in a lactotrope containing small secretory granules. Figure 4. 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. Figure 5. Co-localization of renin (R, 10 nm), PRL (5 nm), and AGT (15 nm). Figure 6. 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. Figure 7. 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. Figure 8. 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
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.

View larger version (164K): [in this window] [in a new window]   Figures 9-11. 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. Figures 9-10. Triple staining: renin (R, 10 nm), ß-LH (LH, 5 nm), and prorenin (pR, 15 nm). Large granulations (arrows) are mainly labeled for ß-LH, whereas the small granules contain ß-LH, renin, and prorenin. Dense structures (curved arrows) are labeled either for both ß-LH and renin ( Figure 9) or for renin only ( Figure 10) (see text). Figure 11. Triple staining: renin (R, 10 nm), ß-LH (LH, 5 nm), and AGT (15 nm). AGT co-localizes with renin and ß-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).

View larger version (138K): [in this window] [in a new window]   Figures 12-20. 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. Figures 12-15. Somatotropes. Figure 12. Double immunostaining for AGT (10 nm, arrows) and growth hormone (GH, 5 nm). Figure 13. 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). Figure 14. 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). Figure 15. 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). Figures 16-18. Corticotropes. Figure 16. Double staining: prorenin (pR, 10 nm; arrows) and ACTH (5 nm). Figure 17. Double staining: AGT (10 nm; arrows) and ACTH (5 nm). Figure 18. Double staining: renin (R, 10 nm; arrow) and ACTH (5 nm). Figures 19-20. Thyrotropes. Figure 19. Double staining: AGT (10 nm; arrows) and ß-TSH (5 nm). Figure 20. Double staining: renin (R, 10 nm; arrows) and ß-TSH (5 nm).

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).

	Discussion

Top Summary Introduction Materials and Methods Results Discussion Literature Cited

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 (Saint-Andre 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 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 (Grimes and Kelly 1992 ) or to the existence in rat lactotropes 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 , Sernia et al. 1997 and Thomas and Sernia 1990 . These authors demonstrated AGT secretion by immunocytochemically 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 gonadotropes 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 eight-fold 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 lysosomal nature of some granulations in rat gonadotropes (Tougard et al. 1974 ), which has also been postulated for lactotropes (Saint-Andre 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 gonadotropes 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 (Deschepper 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-Andre et al. 1989 ) somatotropes. 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 constitutively by transfected AtT-20 cells (Deschepper and Reudelhuber 1990 ) and by in vitro preparations from bovine pituitaries (Colomer et al. 1996 ). Transfection 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 (Ladenheim 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.

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 (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.

	Acknowledgments

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. 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.

Received for publication March 17, 1997; accepted September 3, 1997.

	Literature Cited

Top Summary Introduction Materials and Methods Results Discussion Literature Cited

Arvan P, Castle D (1992) Protein sorting and secretion granule formation in regulated secretory cells. Trends Cell Biol 2:327-331[Medline]

Bendayan M (1982) Double immunocytochemical labeling applying the protein A–gold technique. J Histochem Cytochem 30:81-85[Abstract]

Brechler V, Chu WN, Baxter JD, Thibault G, Reudelhuber TL (1996) A protease processing site is essential for prorenin sorting to the regulated secretory pathway. J Biol Chem 271:20636-20640[Abstract/Free Full Text]

Bouhnik J, Clauser E, Gardes J, Corvol P, Ménard J (1982) Direct radioimmunoassay of rat angiotensinogen and its application to rats in various endocrine states. Clin Sci 62:355-360[Medline]

Campbell DJ, Bouhnik J, Coezy F, Pinet F, Clauser E, Ménard J, Corvol P (1984) Characterization of precursor and secreted forms of rat angiotensinogen. Endocrinology 114:776-785[Abstract]

Campbell DJ, Sernia C, Thomas WG, Oldfield BJ (1991) Immunolocalization of angiotensinogen in rat brain: dependence of neuronal immunoreactivity on method of tissue processing. J Neuroendocrinol 3:653-660

Campbell WG, Jr, Gahnem F, Catanzaro DF, James GD, Camargo MJF, Laragh JH, Sealey JE (1996) Plasma and renal prorenin/renin, renin mRNA, and blood pressure in Dahl salt-sensitive and salt-resistant rats. Hypertension 27:1121-1133[Abstract/Free Full Text]

Colomer V, Kicska GA, Rindler MJ (1996) Secretory granule content proteins and the luminal domains of granule membrane proteins aggregate in vitro at mildly acid pH. J Biol Chem 271:48-55[Abstract/Free Full Text]

Deschepper CF (1991) The renin angiotensin system in the pituitary gland. Trends Endocrinol Metab 2:104-107[Medline]

Deschepper CF, Bouhnik J, Cumin F, Ganong WF (1985) Immunocytochemical localization of the components of the renin-angiotensin system in the pituitary gland of the male rat. Fed Proc 44:1358

Deschepper CF, Crumrine DA, Ganong WF (1986a) Evidence that the gonadotrophs are the likely site of production of angiotensin II in the anterior pituitary of the rat. Endocrinology 119:36-43[Abstract]

Deschepper CF, Ganong WF (1988) Renin and angiotensin in endocrine glands. Frontiers Neuroendocrinol 10:79-98

Deschepper CF, Ganong WF (1991) Distribution of angiotensinogen immunoreactivity in rat anterior pituitary glands. Proc Soc Exp Biol Med 197:304-309[Abstract]

Deschepper CF, Mellon SH, Cumin F, Baxter JD, Ganong WF (1986b) Analysis by immunocytochemistry and in situ hybridization of renin and its mRNA in kidney, testis, adrenal, and pituitary of the rat. Proc Natl Acad Sci USA 83:7552-7556[Abstract/Free Full Text]

Deschepper CF, Reudelhuber TL (1990) Rat angiotensinogen is secreted only constitutively when transfected into AtT-20 cells. Hypertension 16:147-153[Abstract/Free Full Text]

Duhau L, Grassi J, Grouselle D, Enjalbert A, Grognet JM (1991) An enzyme immunoassay for rat prolactin: application to the determination of plasma levels. J Immunoassay 12:233-250[Medline]

Fritz LC, Haidar MA, Arfsten AE, Schilling JW, Carilli C, Shine J, Baxter JD, Reudelhuber TL (1987) Human renin is correctly processed and targeted to the regulated secretory pathway in mouse pituitary AtT-20 cells. J Biol Chem 262:12409-12412[Abstract/Free Full Text]

Galen FX, Devaux C, Atlas S, Guyenne T, Ménard J, Corvol P, Simon D, Cazaubon C, Richer P, Badouaille G, Richaud JP, Gros P, Pau B (1984) New monoclonal antibodies directed against human renin. Powerful tools for the investigation of the renin system. J Clin Invest 74:723-735

Ganong WF (1993) Blood, pituitary and brain renin-angiotensin systems and regulation of secretion of anterior pituitary gland. Frontiers Neuroendocrinol 14:233-249[Medline]

Ganong WF (1994) Origin of the angiotensin II secreted by cells. Proc Soc Exp Biol Med 205:213-219[Abstract]

Ganong WF (1995) Reproduction and the renin-angiotensin system. Neurosci Biobehav Rev 19:241-250[Medline]

Ganong WF, Deschepper CF, Steele MK, Intebi A (1989) Renin-angiotensin system in the anterior pituitary of the rat. Am J Hypertens 2:320-322[Medline]

Grimes M, Kelly RB (1992) Intermediates in the constitutive and regulated secretory pathways released in vitro from semi-intact cells. J Cell Biol 117:539-549[Abstract/Free Full Text]

Hackenthal E, Paul M, Ganten D, Taugner R (1990) Morphology, physiology, and molecular biology of renin secretion. Physiol Rev 70:1067-1116[Free Full Text]

Hellmann W, Suzuki F, Ohkubo H, Nakanishi S, Ludwig G, Ganten D (1988) Angiotensinogen gene expression in extrahepatic rat tissues: application of a solution hybridization assay. Naunyn Schmiedebergs Arch Pharmacol 338:327-331[Medline]

Hurbain-Kosmath J, Bérault A, Noël N, Polkowska J, Bohin A, Jutisz M, Leiter EH, Beamer WG, Bedigian HG, Davisson MT, Harrison DE (1990) Gonadotropes in a novel rat pituitary tumor cell line, RC-4B/C. Establishment and partial characterization of the cell line. In Vitro Cell Dev Biol 26:431-440[Medline]

Kettani S, Beldent V, Rousselet MC, Ronco P, Verroust P, Saint-André JP (1991) Presence of renin, angiotensinogen, angiotensin II in the lamb anterior pituitary gland: immunocytochemical study after cryoultramicrotomy. Histochemistry 95:561-566[Medline]

Ladenheim RG, Seidah N, Lutfalla G, Rougeon F (1989) Stable and transient expression of mouse submaxillary gland renin cDNA in AtT-20 cells: proteolytic processing and secretory pathways. FEBS Lett 245:70-74[Medline]

Mukai M, Torikata C, Hirose S, Murakami K, Kageyama K (1984) Biochemical and immunohistochemical localization of renin in human pituitary. Lab Invest 51:425-428[Medline]

Naruse K, Naruse M, Obana K, Demura R, Demura H, Inagami T, Shizume K (1986) Renin in the rat pituitary coexists with angiotensin II and depends on testosterone. Endocrinology 118:2470-2476[Abstract]

Naruse K, Takii Y, Inagami T (1981) Immunohistochemical localization of renin in luteinizing hormone-producing cells of rat pituitary. Proc Natl Acad Sci USA 78:7579-7583[Abstract/Free Full Text]

Newman GR (1987) Use and abuse of LR White. Histochem J 19:118-120[Medline]

Ozawa H, Picart R, Barret A, Tougard C (1994) Heterogeneity in the pattern of distribution of the specific hormonal product and secretogranins within the secretory granules of rat prolactin cells. J Histochem Cytochem 42:1097-1107[Abstract]

Phillips MI, Speakman EA, Kimura B (1993) Tissue renin-angiotensin systems. In Raizada MK, Phillips MI, Sumners C, eds. Cellular and Molecular Biology of the Renin-Angiotensin System. Boca Raton, FL, CRC Press, 97-130

Pratt RE, Flynn JA, Hobart PM, Paul M, Dzau VJ (1988) Different secretory pathways of renin from mouse cells transfected with the human renin gene. J Biol Chem 263:3137-3141[Abstract/Free Full Text]

Reudelhuber TL, Mercure C, Théroux LL, Chu WN, Baxter JD, Seidah NG (1993) Subcellular sorting and processing of prorenin. In Raizada MK, Phillips MI, Sumners C, eds. Cellular and Molecular Biology of the Renin-Angiotensin System. Boca Raton, FL, CRC Press, 3-31

Rousselet MC, Beldent V, Pinet F, Galland F, Rohmer V, Bigorgne JC, Saint-André JP (1990) Immunocytochemical and biochemical evidence of renin in human lactotropic cell cultures. Lab Invest 63:370-376[Medline]

Saavedra JM (1992) Brain and pituitary angiotensin. Endocrine Rev 13:329-380[Medline]

Saint-André JP, Rhomer V, Pinet F, Rousselet MC, Bigorgne JC, Corvol P (1989) Renin and cathepsin B in human pituitary lactotroph cells. An ultra-structural study. Histochemistry 91:291-297[Medline]

Saint-André JP, Rohmer V, Alhenc–Gelas F, Ménard J, Bigorgne JC, Corvol P (1986) Presence of renin, angiotensinogen and converting enzyme in human pituitary lactotroph cells and prolactin adenomas. J Clin Endocrinol Metab 63:213-237

Sernia C (1995) Location and secretion of brain angiotensinogen. Regul Pept 57:1-18[Medline]

Sernia C, Shinkel TA, Thomas WG, Ho KKY, Lincoln D (1992) Angiotensinogen secretion by single pituitary cells: detection by a reverse hemolytic plaque assay and cell identification by immunocytochemistry. Neuroendocrinology 55:308-316[Medline]

Sernia C, Zeng T, Kerr D, Wyse B (1997) Novel perspectives on pituitary and brain angiotensinogen. Frontiers Neuroendocrinol 18:174-208[Medline]

Steele MK, Brownfield MS, Ganong WF (1982) Immunocytochemical localization of angiotensin immunoreactivity in gonadotrops and lactotrops in the rat anterior pituitary gland. Neuroendocrinology 35:155-158[Medline]

Taugner R, Whalley A, Angermuller S, Buhrle CP, Hackenthal E (1985) Are the renin-containing granules of juxtaglomerular epithelioid cells modified lysosomes? Cell Tissue Res 239:575-587[Medline]

Thomas WG, Sernia C (1990) Immunocytochemical localization of angiotensinogen and angiotensin II in the rat pituitary. J Neuroendocrinol 2:297-304

Tougard C, Picart R, Tixier–Vidal A, Kerdelhué B, Jutisz M (1974) In situ immunochemical staining of gonadotropic cells in primary cultures of rat anterior pituitary cells with the peroxidase labelled antibody technique. A light and electron microscopic study. In Wisse E, Daems WT, Molenaar I, van Djuin P, eds. Electron Microscopy and Cytochemistry. Amsterdam, North–Holland, 163-166

Vila–Porcile E, Barret A (1996) Structural and functional differences between prolactin cells from the inner and outer zones of the male rat anterior pituitary. Cell Tissue Res 284:247-249[Medline]

CiteULike    Complore    Connotea    Del.icio.us    Digg    Reddit    Technorati    What's this?

This article has been cited by other articles:

				M. Paul, A. Poyan Mehr, and R. Kreutz Physiology of local Renin-Angiotensin systems. Physiol Rev, July 1, 2006; 86(3): 747 - 803. [Abstract] [Full Text] [PDF]

				M. Landry, E. Vila-Porcile, and A. Calas Immunogold Detection of Co-localized Neuropeptides: Methodological Aspects J. Histochem. Cytochem., May 1, 2004; 52(5): 617 - 628. [Abstract] [Full Text] [PDF]

				E. Vila–Porcile, A. Barret, and P. Corvol Secretion of Renin-Angiotensin System (RAS) Components by Normal and Tumoral Lactotropes: A Comparative Study Using Reverse Hemolytic Plaque Assay (RHPA) and Immunoelectron Microscopy J. Histochem. Cytochem., December 1, 2000; 48(12): 1691 - 1704. [Abstract] [Full Text]

				Y. Kusaka, R. A. Kelly, G. H. Williams, and I. Kifor Coronary microvascular endothelial cells cosecrete angiotensin II and endothelin-1 via a regulated pathway Am J Physiol Heart Circ Physiol, September 1, 2000; 279(3): H1087 - H1096. [Abstract] [Full Text] [PDF]

				S. Vidal, S. M. Cohen, E. Horvath, K. Kovacs, B. W. Scheithauer, B. G. Burguera, and R. V. Lloyd Subcellular Localization of Leptin in Non-tumorous and Adenomatous Human Pituitaries: An Immuno-ultrastructural Study J. Histochem. Cytochem., August 1, 2000; 48(8): 1147 - 1152. [Abstract] [Full Text]

This Article Abstract Full Text (PDF) Alert me when this article is cited Alert me if a correction is posted Citation Map Services Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Citing Articles Citing Articles via HighWire Citing Articles via Google Scholar Google Scholar Articles by Vila–Porcile, E. Articles by Corvol, P. Search for Related Content PubMed PubMed Citation Articles by Vila–Porcile, E. Articles by Corvol, P. Social Bookmarking                      What's this?

Guidelines | Subscriptions | About | exPRESS - Current - Archive | Business Information | Contact