Secretion of renin-angiotensin system (RAS) components by normal and tumoral lactotropes. A comparative study using reverse hemolytic plaque assay (RHPA) and immunoelectron microscopy.

Immunodetection of renin-angiotensin system (RAS) components indicates that there is a local RAS in anterior pituitary cells, particularly in lactotropes. We have attempted to determine if RAS molecules are secreted by lactotropes and the secretory pathways and intracellular sites of maturation. We investigated the secretory activity of individual lactotropes, using the reverse hemolytic plaque assay (RHPA), with GH3B6 tumor cells and normal male rat pituitary cells. We also determined the subcellular distributions of RAS components in these cells. Both tumor and normal cells secreted angiotensinogen, prorenin, renin, angiotensin I, angiotensin-converting enzyme, and angiotensin II, although at different levels. The percentage of secretory cells was generally higher in tumor lactotropes than in normal cells. The subcellular distribution of RAS components obtained by immunoperoxidase was very similar in both cell types, although the intensities of immunoreactivity differed. Cleaved and uncleaved components were found in rough endoplasmic reticulum (RER), Golgi saccules, and secretory granules, all compartments of the secretory pathway. The cleaved components in the RER suggest the existence of early maturation, whereas the presence of uncleaved products in the secretory granules of normal lactotropes might indicate late maturation sites.

Effects of centrally administered galanin (1-16) on galanin expression in the rat hypothalamus.

In the rat hypothalamic magnocellular neurons, galanin coexists with vasopressin and might be involved in hydro-osmotic regulation. In the present study, we investigated the ability of galanin to also regulate the osmotically stimulated expression of galanin itself in hypothalamic magnocellular neurons. Ten minutes after galanin injection, galanin mRNA rate decreased in salt-loaded rats whereas the level of galanin immunoreactivity increased. Both effects were suppressed by the injection of a galanin antagonist together with galanin. Moreover, electron microscope studies demonstrated synaptic contacts between galanin-containing fibers and magnocellular neurons. Galanin may exert inhibitory roles in the regulation of magnocellular neurons. However, galanin and vasopressin expression displayed differences upon galanin injection. Possible mechanisms underlying these discrepancies are further discussed.

Angiotensinogen, prorenin, and renin are Co-localized in the secretory granules of all glandular cells of the rat anterior pituitary: an immunoultrastructural study.

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.

Structural and functional differences between prolactin cells from the inner and outer zones of the male rat anterior pituitary.

The aim of this study was to compare the ultrastructure of prolactin cells in the inner and outer zones of the male rat anterior pituitary, and to relate their morphological features to their secretory activity, by means of standard and ultrastructural reverse hemolytic plaque assays (RHPA). The immuno-ultrastructural study showed that in the inner pituitary small-granulated cells represented 52% of the prolactin cells, there being only 5% with large granules, whereas the prolactin cells with large granules accounted for 52% in the outer zone, with only 7% being small-granulated. Percentages of cells with intermediate-sized granules were 43% and 41%, respectively. Analysis of RHPA data revealed that, under basal conditions, prolactin cells secreted more actively in the inner zone than in the outer zone. Stimulation with thyrotropin-releasing hormone or KCl treatment increased the percentage of secretors and the sizes of hemolytic plaques in both zones. However, in response to thyrotropin-releasing hormone, the increase in number of secretors was always higher in the outer zone, whereas the enlargement of plaque sizes was greater for the "inner" cells. These findings are in favor of the small-granulated cells, which predominate in the inner zone, being in a stage of active secretion and responsiveness.

Aggregation of a subpopulation of vimentin filaments in cultured human skin fibroblasts derived from patients with giant axonal neuropathy.

Giant axonal neuropathy (GAN) is a generalized disorder of intermediate filament networks which results in the formation of an ovoid aggregate in a large variety of cell types. We investigated the cytoskeletal organization of cultured skin fibroblasts derived from three GAN patients by indirect immunofluorescence, confocal, and electron microscopy. Whereas the organization of microfilaments seemed normal, the microtubule network appeared disorganized and tangled. The organization of the intermediate filament network, composed of vimentin, was probed with three antibodies directed against different epitopes: two vimentin-specific antibodies, a monoclonal antibody (mAb V9) and a polyclonal antibody, and a serum specific for all type III IFPs (PI serum). These experiments showed that 20% of cultured skin fibroblasts from GAN patients have a vimentin aggregate composed of densely packed filaments which coexists with a well-organized vimentin network. After depolymerization of microtubules with nocodazole, all fibroblasts from GAN patients contained a vimentin aggregate which seemed to arise from a subpopulation of vimentin filaments normally integrated in the vimentin network. Such aggregates were never observed in any condition in control fibroblasts. Moreover, the ultrastructural analysis of GAN cells revealed the presence of swollen mitochondria. We suggest that GAN may be due to a defect in a factor which stabilizes cytoplasmic intermediate filament networks, and we speculate on its identification and properties.

Adaptation of the reverse hemolytic plaque assay to electron microscopy: a study of the individual secretory activity in prolactin cell subpopulations.

We used the reverse hemolytic plaque assay (RHPA), a method for detecting secretion by single cells, to demonstrate functional heterogeneity among prolactin (PRL) cells of rat anterior pituitary at the light microscopic (LM) level. We attempted to adapt RHPA for electron microscopy (EM) to define the relationships between fine structure and secretory activity in individual PRL cells. A major modification of the technique, intended to improve preservation of ultrastructure, was allowing the cells to recover after their enzymatic dispersion from the pituitary tissue, through a brief (3-4 hr) culture step before the assay. Adaptation for EM was achieved by the use of plastic slides for construction of specialized Cunningham chambers, permitting application of all the EM procedures (flat embedding, punching of selected areas) usually employed for cultured pituitary monolayers. Moreover, immunocytochemical pre- and post-embedding methods were also applied for cell identification and study of subcellular hormone distribution. Such a modified RHPA enabled us to analyze the ultrastructure of plaque-forming cells surrounded by their companion red blood cell ghosts. The first results with EM RHPA showed that under basal conditions a subpopulation of PRL cells containing small granules (150-200 nm) was actively secreting, whereas PRL cells with large (300-600 nm) and irregular granules appeared to be stimulated by thyrotropin-releasing hormone or KCl. The EM RHPA technique described here might receive more general application and could be utilized for study of many other secretory systems.

Immunolocalization of laminin, heparan-sulfate proteoglycan, entactin and type IV collagen in the rat anterior pituitary. II. An in vitro study on primary cultures.

The distribution of four basement membrane components: laminin (LAM), type IV collagen (Coll. IV), heparan-sulfate proteoglycan (HSPG), and entactin (ENT), was studied by immunocytochemistry in primary cultures of adult rat anterior pituitaries. In such cultures, the pituitary cells are deprived of their normal environment of adjacent cells and basement membranes (BM), and of the connectivo-vascular system of the hypophysis. In this dissociated system, pituitary cells grow as small clusters upon a monolayer of fibroblasts. LAM was found highly expressed in endocrine and in folliculo-stellate cells. Very small amounts of Coll. IV, but neither HSPG nor ENT, could be detected in endocrine cells. In contrast, in fibroblasts, very large amounts of Coll. IV, HSPG, and ENT, and a lower quantity of LAM were detected. At the ultrastructural level, the immunoreactive components present within the cells were located in the subcellular compartments involved in the elaboration of exported products. In addition to that intracellular distribution, the four constituents were observed in an extracellular matrix which appeared between the cultured cells, either as an amorphous material, or as a more or less dense reticular network, weakly stained with anti-LAM, but strongly stained with the other antibodies. Thus, the present immunocytochemical data support the implication of pituitary endocrine cells, at least for LAM secretion, in the elaboration of a novel extracellular matrix in primary cultures. In addition, a cooperation with non-endocrine cells seemed to be required for the production of the four BM components.

Immunolocalization of laminin, heparan-sulfate proteoglycan, entactin, and type IV collagen in the rat anterior pituitary. I. An in vivo study.

The distribution of three components of basement membranes (BM): heparan-sulfate proteoglycan (HSPG), entactin (ENT), and type IV collagen (Coll. IV), was studied in the adult rat anterior pituitary and compared to the distribution of laminin (LAM) described in an earlier report (Vila-Porcile et al., 1987, J. Histochem. Cytochem., 35, 287). Several immunocytochemical methods were applied at both light and electron microscope levels. The three components were detected in all the pituitary BM and in endothelial and perivascular connective cells, as previously observed for LAM. In contrast to the prominent labeling previously observed for LAM within epithelial cells, however, only a faint immunoreaction could be detected for the other components, with nonetheless a discrete signal for Coll. IV. These findings indicate that 1) the four studied components are present in all the BM of the rat anterior pituitary; 2) pituitary endocrine cells contain LAM, and to a lesser extent Coll. IV, and thus could participate to the elaboration of the BM; and 3) the presence of the four components in non-epithelial cells also suggests their cooperation in BM building. The questions of the turnover and intracellular pathways of each of these components were addressed but remain unanswered.

Pituitary pathology in Cushing's disease. Histology and morphometry of pituitary tissues removed through microsurgery.

58 cases of Cushing's disease were studied after selective transsphenoidal surgery, by means of histology, immunocytochemistry and morphometry. The qualitative and quantitative results, related to the clinical follow-up of the patients indicated that: 1) when a well-defined tumor was found (62% of the patients), successful outcomes occurred in 88% of the cases, 2) when a tumor could not be found, successful outcomes amounted to only 33% of the cases, a rate that might be related to a surgical exploration of the basophil cell zone "invading" the neural lobe, thus questioning a role of this zone, at least in some Cushing's diseases.

Transsphenoidal pituitary surgery for the treatment of Cushing's disease: results in 64 patients and long term follow-up studies.

Transsphenoidal pituitary surgery was performed in 64 patients with Cushing's disease in search of a corticotroph adenoma. In 4 patients, profuse local bleeding prevented completion of the exploration. Of the 60 patients who had an adequate exploration, 4 could not be followed after surgery. Short term assessment of the surgical outcome (3-6 months postoperatively) was performed on 60 patients, including the 4 who had incomplete pituitary exploration. Forty-two patients (70%) were judged as immediate successes [urinary cortisol excretion, less than 90 micrograms (less than 248 nmol)/day]; the mean urinary cortisol excretion and plasma ACTH level fell from 463 +/- 70 (+/- SE) to 26.7 +/- 3.6 micrograms/day (1277 +/- 193 to 74 +/- 10 nmol/day; n = 33) and from 111 +/- 33 to 36 +/- 14 pg/mL (24 +/- 7 to 8 +/- 3 pmol/L; n = 23) in patients who had both measurements pre- and postoperatively. Eighteen patients (30%) were judged as immediate failures; neither urinary cortisol excretion nor plasma ACTH levels changed significantly in patients who had both measurements pre- and postoperatively. The preoperative epidemiological, clinical, hormonal, and radiological characteristics of the 2 groups were similar. Histological examination of pituitary fragments removed in 58 of the 60 patients evaluated postoperatively revealed the presence of tumoral tissue in a higher percentage of patients in the immediate success group (72%) than in the immediate failure group (24%; P less than 0.01). The 42 patients in the immediate success group were followed from 6 months to 7 yr (median, 2 yr); 6 patients had recurrences from 2-3 yr after operation. Actuarial analysis indicates that the probability of a patient remaining well 6 yr after surgery is 72 +/- 20% (95% confidence limit). Most of the patients in the immediate success group had transient ACTH deficiency preceding a progressive return to normal pituitary-adrenal function.

Subcellular distribution of laminin and prolactin in stimulated and blocked prolactin cells in the pituitary of lactating rats.

Laminin (LAM), a glycoprotein component of basement membranes, has been previously detected within several subcellular compartments of prolactin (PRL) cells in the pituitary gland. The present work was aimed at comparing the subcellular localization of PRL, a specific secretory product, with that of LAM, in relation to the secretory activity of PRL cells. LAM and PRL were located in parallel, by ultrastructural immunocytochemistry, in PRL cells of lactating female Wistar rats, either stimulated by suckling, or blocked by weaning, or reactivated by suckle following short-term weaning. Variations in physiological conditions were correlated with a redistribution of PRL immunoreactivity within morphologically modified compartments. The Golgi apparatus became hypertrophied, and PRL impressively accumulated within saccules of the Golgi stacks of blocked cells. On the contrary, no apparent changes occurred in LAM distribution, at least at the Golgi level. Only a slight increase of LAM immunoreactivity was observed in rough endoplasmic reticulum after a long weaning period. PRL could be detected in most of the secretory granules and particularly in forming elements, whereas LAM was observable at the peripheral edge of some mature granules. Such a labeling was not markedly influenced by the physiological state. The prominent structures, indicative of crinophagic activity, characteristic of blocked cells, contained masses of dense material, which were always immunopositive with antibodies to PRL, but never to LAM. These observations could suggest that, in PRL cells, intracellular transport and exportation of LAM are controlled by mechanisms independent from those involved in the regulation of PRL secretion.

Cellular and subcellular distribution of laminin in adult rat anterior pituitary.

Following the immunodetection of laminin in basal laminae and within some glandular cells of adult rat pituitary (Tougard et al., 1985: In Vitro 21:57), the present work had a double purpose: (a) adjustment of optimal conditions to detect laminin at both the cellular and subcellular level with an immunoperoxidase pre-embedding procedure, and (b) identification of the hormonal specificity of the laminin-containing cells, based on development of a "two-step" method combining the pre- and post-embedding approaches. In addition to extracellular localizations (basal laminae and connective tissue compartment), laminin was detected intracellularly within membrane-bounded compartments in all endocrine cell types and in the folliculo-stellate cells. It was found in rough endoplasmic reticulum cisternae, in a few Golgi saccules, and in vesicles or vacuoles. Labeled secretory granules were observed in gonadotrope, thyrotrope, and corticotrope cells but were very scarce in prolactin cells and absent in somatotrope cells. In addition, exocytotic profiles of labeled granules were frequent, suggesting a granular pathway for laminin export. Labeled vesicles, heterogeneous in size and shape, were observed mostly in prolactin cells. They might represent either a vesicular pathway for laminin export or an endocytic route for membrane-bounded laminin. These results bring complementary data to the concept that epithelial cells elaborate their basement membranes (Pierce and Nakane, 1967: Lab Invest 17:499). Moreover, the subcellular distribution of laminin within organelles involved in the biosynthesis, transport, and even in the storage of secretory products suggests that laminin might be exported by glandular pituitary cells according to different pathways.

[Cellular localization of peptides derived from pro-opiocortin in the normal human adenohypophysis and in tumors of Cushing's disease]. / Localisations cellulaires des peptides dérivés de la propiocortine dans l'adénohypophyse humaine normale et dans les tumeurs de la maladie de Cushing.

As early as 1932, basophil cells have been considered to be involved in the corticotropic and melanotropic functions due to their proliferation during Cushing's disease. Later on, opposite opinions were held and other cells suggested to be implied in the tumor process: chromophobe, acidophil or even follicular cells. Moreover, new categories of basophil cells have been evidenced through sophisticated staining methods, in animal and human pituitaries. At the same time, the prevalent dogma 'one hormone-one cell' raised the problem of the search for both a corticotroph and a melanotroph in the human pituitary, which is devoid of an intermediate lobe. Since 1960, immunocytochemistry became a reliable method and simultaneously new peptides have been found. However, these peptides share common sequences of amino acids, thus raising questions as to the specificity of the antisera. Most authors mention the reaction to the basophil cells previously considered as melanotrophs and called beta or R [here beta(R)]. These large rounded cells, showing red granules after Alcian blue-PAS staining, are mostly located in the median area of the anterior lobe, in the cystiform zone and in cell cords invading the neural lobe. In the fetal pituitary, reactivity is observed either in some differentiated beta(R) cells or, earlier, in chromophobe cells. The question is now raised whether one cell may contain several peptides and, in this case, whether the different peptides are carried by the same granule. Technical procedures using numerous antisera on serial sections have shown associations at the level of a given cell (and of a given granule under the electron microscope). However, dissociations are also to be noticed, as far as ACTH and beta-MSH (adult) and more particularly alpha-MSH (adult and fetus) are concerned.