Heterogeneity of pituitary gonadotrope cells in male rats.

Mammalian gonadotrope (Gn-) cells producing FSH and LH show great variability in their cytological characteristics. To gain a closer view of the corresponding phenotypes, Gn-cells of male rats were investigated by various methods including immunocytochemistry on serial semithin sections, quantitative immunocytochemistry, three-dimensional reconstructions, sequential semithin-/thin-section technique, routine electron microscopy, and immuno-electron microscopy. Gn-cells were immunostained with FSH and LH antisera and with antibodies raised against synthetic N- and C-terminal sequences of chromogranin A (CgA) and secretogranin II (SgII), and four phenotypes of Gn-cells could be identified. The great majority of Gn-cells were densely immunoreactive for both gonadotropins and both chromogranins and accordingly classified as "typical" gonadotropes; a second type was less immunoreactive for LH and therefore was termed "LH-poor". The two remaining phenotypes showed ultrastructures suggesting an increased synthetic activity. One of them was characterized as a "FSH/CgA-poor" gonadotrope. The other, also poor in CgA, morphologically corresponded to "signet-ring" cells previously known only in gonadectomized animals. The two types of secretory granules present in male rat Gn-cells showed a strict segregation of chromogranin immunoreactivities: CgA was confined to the large, less opaque granules, whereas SgII, to the small, electron-dense granules. Consequently the two "CgA-poor" Gn-cell subtypes exhibited only a very few large-sized secretory granules. A correlation was suggested between high cellular activity and a decrease in CgA in Gn-cells. The reasons for the morphological and functional heterogeneity of Gn-cells remain enigmatic. Presumably, they either differ in the susceptibility for GnRH and testosterone or are differently regulated by paracrine factors.

Bronchiolar nonciliated secretory (Clara) cells: source of guanylin in the mammalian lung.

The peptide guanylin, which has recently been isolated from the intestine, is involved in the regulation of fluid secretion in the intestinal epithelium by activation of guanylate cyclase C, the putative guanylin receptor. Since the latter protein is also expressed in airway epithelia, we investigated the lung of three mammalian species for the presence and cellular localization of guanylin by immunoblot (Western blot) analyses and light and electron microscopical immunocytochemistry. In Western blots of bovine, guinea pig, and rat lung extracts, three different guanylin antisera directed against the midportion and against the C terminus of the precursor molecule identified a peptide band corresponding to the apparent molecular mass of guanylin. Localization studies in the lung revealed that guanylin is exclusively confined to nonciliated secretory (Clara) cells in the lining of distal conducting airways. The presence of guanylin in the lung and particularly its specific localization to Clara cells indicate that these cells may play a pivotal role in the local (paracrine) regulation of electrolyte/water transport in airway epithelia.

Enterochromaffin cells of the digestive system: cellular source of guanylin, a guanylate cyclase-activating peptide.

Guanylin, a bioactive peptide, has recently been isolated from the intestine; this peptide activates intestinal guanylate cyclase (i.e., guanylate cyclase C) and thus is potentially involved in the regulation of water/electrolyte transport in the gastrointestinal mucosa. As yet, the cells involved in synthesis, storage, or secretion of guanylin have not been identified by immunocytochemistry. We raised antisera against guanylin and investigated the entire gastrointestinal tract of guinea pigs by light and electron microscopical immunocytochemistry. Extracts of various intestinal segments and plasma analyzed on a Western blot revealed a peptide band corresponding to the molecular mass of guanylin. Localization studies in the entire digestive tract showed that guanylin is exclusively confined to enterochromaffin (EC) cells. Remarkably, most EC cells contacted the gut lumen by cell processes that were highly immunoreactive for guanylin. In addition to the well known secretion in an endocrine fashion, EC cells by circumstantial evidence may release guanylin into the gut lumen to activate guanylate cyclase C that is immediately located on the brush border of adjacent enterocytes. The unique localization of guanylin in EC cells may indicate that these cells are involved in the regulation of fluid secretion in the gastrointestinal mucous membrane.

Chromostatin, a chromogranin A-derived bioactive peptide, is present in human pancreatic insulin (beta) cells.

Chromogranin A (CGA) is a secretory protein present in the adrenal medulla and in a variety of endocrine organs. This protein may serve as precursor for pancreastatin (PST) and for other biologically active peptides. Recently, chromostatin (CST), a CGA derivative, has been identified that possesses high biological activity. The cellular distribution of CST in various endocrine organs is completely unknown. Using immunohistochemistry on plastic sections, we investigated the occurrence and cellular distribution of CST, PST, and CGA in human endocrine pancreas of healthy and diseased states and in the adrenal medulla. In the normal and diabetic pancreas, CST immunoreactivity was localized exclusively in beta cells, which were mostly unreactive for PST and CGA. Both latter peptides were confined mainly to glucagon (alpha) cells. Insulinoma cells displayed strong insulin, PST, and CGA immunoreactivities, but they were faintly immunoreactive for CST or unreactive. Adrenal chromaffin cells exhibited strong immunoreactivity for CGA but lacked CST and PST immunoreactivities. Based on the peculiar distributive pattern of CST, PST, and CGA, we suggest that CGA is differentially processed in chromaffin and islet tissues and in insulinoma cells. The unique cellular localization of CST in the endocrine pancreas of normal and pathological conditions may indicate that CST is involved in beta-cell function.

Synaptophysin--a common constituent of presumptive secretory microvesicles in the mammalian pinealocyte: a study of rat and gerbil pineal glands.

Recent studies have established that pinealocytes of the mammalian pineal gland contain marker molecules of neuroendocrine cells or paraneurons like the synaptic vesicle-associated protein synaptophysin (p38). The objective of this study was to identify the subcellular synaptophysin-positive compartment and to characterize in detail the intracellular distribution of this protein in rat and gerbil pinealocytes. An analysis of serial semithin sections of plastic-embedded pineals immunostained for synaptophysin, including computer-assisted optical density measurements of synaptophysin immunoreactivities, demonstrated unequivocally that synaptophysin was highly concentrated in dilated process terminals of the pinealocytes. More than 75% of these process terminals were found to border or lie within the pericapillary space. At the ultrastructural level, they contained accumulations of small clear vesicles of variable size that turned out to be the site of synaptophysin immunoreactivity when immunogold staining was performed. In addition, microvesicles surrounding synaptic ribbons were also immunolabeled. Hence, the pinealocyte is the first neuroendocrine cell type that has now been shown to concentrate synaptophysin-positive microvesicles in perivascular process endings. This observation lends strong support to the hypothesis that small clear vesicles in neuroendocrine cells in general, and in pinealocytes in particular, serve secretory functions. The quantitative analysis of completely sectioned process endings revealed that the microvesicles outnumber by far the amount of dense core vesicles and therefore cannot arise by endocytosis of dense core vesicle membranes. Thus, small synaptic-like vesicles probably constitute an independent secretory pathway of the paraneuronal pinealocytes. In the present study, we could also establish the absence of immunoreactivity for synapsin I (belonging to a family of neuron-specific nerve terminal phosphoproteins) from pinealocytes. Synapsin I immunoreactivity was only detectable in intrapineal nerve terminals and varicosities. Taken together, the immunostaining patterns of the pineal gland obtained with antibodies directed against synaptic vesicle-associated proteins render the mammalian pinealocyte a very special type of neuroendocrine cell or paraneuron rather than a "classic" neuron.

Serotonin storage and chromogranins: an experimental study in rat gastric endocrine cells.

Chromogranins (Cg) and secretogranins (Sg) are acidic proteins localized in the secretory granules of a large variety of endocrine cells collectively named APUD cells (amine precursor uptake and decarboxylation). To examine the possible function of Cg/Sg as amine storage proteins, enteroendocrine cells of the rat gastric antral mucosa, i.e., serotonin-containing enterochromaffin (EC)-cells, gastrin (G)-, and somatostatin (D)-cells, were investigated immunohistochemically in serial semi-thin sections of controls and after intervention in serotonin synthesis. CgA and CgB immunoreactivity was determined semiquantitatively by optical density measurements. Experiments included inhibition of serotonin synthesis by p-chlorophenylalanine (pCPA), exogenous application of the serotonin precursor 5-hydroxytryptophan (5-HTP), and a combination of both treatments. The cellular distribution of Cg and the density of its immunoreactivity were closely related to the primary content of serotonin and the ability to store serotonin after 5-HTP application. Thus, Cg may act as amine-binding proteins in enteroendocrine cells, binding most probably being due to ionic interactions between Cg and the biogenic amines. EC- and G-cells, however, differed in their amine-handling properties and in the response of their Cg immunoreactivity after intervention in serotonin synthesis. We conclude, therefore, that the physiological function of Cg as amine storage proteins is restricted to endocrine cells with an endogenous content of amines. In other endocrine cells, exhibiting only a potential amine production, APUD may be considered as a kind of supravital staining without physiological significance.

Mutual relationships between chromogranins A and B and gastrin in individual gastrin cells.

The chromogranins A and B (CgA and CgB, respectively), originally detected in the adrenal medulla, are present in various endocrine organs. Remarkably, their immunoreactivities vary among different endocrine cell types and also within a given endocrine cell population. With densitometric techniques at the cellular level, individual gastrin cells (n = 318) from guinea pig antral mucosa were studied to measure their content of immunoreactive CgA, CgB, and gastrin. The composition of these secretory proteins in individual gastrin cells varied considerably but with predictable components. Statistical evaluation of the data showed that immunoreactivities for gastrin and CgA correlated negatively in these cells; CgA and CgB immunoreactivities also correlated inversely. On the other hand, immunoreactivities for gastrin and CgB exhibited a high positive correlation. The mutual relationships between gastrin, CgA, and CgB suggest that under physiological conditions biosynthetic pathways of these secretory constituents are linked to each other in individual gastrin cells.

The microanatomy of canine islets of Langerhans: implications for intra-islet regulation.

In recent years models for the internal ("intra-islet") regulation of hormone secretion have been proposed to explain how different islet cells might regulate each other by means of their respective secretory peptides. Models that emphasize the importance of a directed intra-islet blood flow and sequence of perfusion of islet cells rely on a certain type of islet microanatomy and vascular supply. The experimental studies underlying these models have partly been performed in dogs. To extend the incomplete morphological knowledge of the canine endocrine pancreas both canine islets of Langerhans and extrainsular cells have been analysed in immunostained serial semithin (0.5 microns) sections. In addition to their occurrence within islets of Langerhans, all endocrine cell types are also found at extrainsular sites (about 9% of all endocrine cells) where they are distributed in different quantities among the epithelial lining of exocrine acini or excretory ducts and the connective tissue. There are continuous transitions from single extrainsular cells to small mono- and polycellular cell groups to islets. In a comprehensive analysis of whole islets, including computer-assisted three-dimensional reconstructions, the size, shape and vascularization of the islets as well as their cellular composition and the microtopology of islet cells have been studied. We have found marked intra- and inter-islet heterogeneities of the parameters investigated that are not compatible with concepts of a uniform and directed vascular perfusion of the various islet cell populations. Instead, their paracrine regulation may occur primarily via hormonal secretion into the intercellular spaces or vascular hormonal delivery to adjacent cells.

Chromogranins in mammalian GEP endocrine cells: their distribution and interrelations with co-stored amines and peptides.

Chromogranins (Cg) are large acidic proteins, co-stored with amines or peptides in the secretory granules of a wide variety of paraneurons. By immunohistochemistry performed on serial semithin sections, the distribution of Cg in the gastroenteropancreatic (GEP) system was analyzed in the endocrine pancreas of 10 mammalian species and in enteroendocrine cells of 6 species. Of the Cg families, CgA is primarily localized in GEP endocrine cells. CgA was regularly present in amine containing cells (EC and EC-like cells). In 11 other endocrine cell types, CgA-immunoreactivities showed inter-species and intra-species variations in their distribution or in the intensity of staining. However, no endocrine cell type lacked CgA-immunoreactivities in all species investigated. Findings from extended studies indicate that CgA is involved in the intracellular storage of resident amines, and in the storage of newly formed amines after exogenous application of the corresponding precursors. Finally, densitometric findings on possible reciprocal relationships between CgA- and peptide-immunoreactivities suggest that CgA also participates in the intragranular storage of peptide hormones in certain GEP endocrine cells.

On the formation and degradation of melanosomes in smooth muscle cells: electron microscopic investigation on the m. sphincter pupillae of the rat.

The electron microscopic investigation of the m. sphincter pupillae of adult black hooded rats showed the presence of melanosomes in the smooth muscle cells. In shape and size the melanosomes were like those of the iridial epithelium. In addition, premelanosomes and tyrosinase activity were observed as well as melanosomes with disintegrated content and acid phosphatase activity. The data suggest that the smooth muscle cells of the m. sphincter pupillae are capable of the formation and degradation of melanosomes.

Light and electron microscopic study on the osteoclastic phagocytosis of cells in the rat.

By means of micromorphological and cytochemical investigations of the tibia metaphyses of growing rats the phagocytosis of cells by multinucleated osteoclasts could be demonstrated. On the average 1.9% of osteoclasts exhibited included cells. With very few exceptions the phagocytosed cells belonged to the monocyte/macrophage lineage, as shown by evidence from the ultrastructure. After the animals were treated with parathyroid hormone the number of osteoclasts increased significantly, but not, however, the number of osteoclasts with a cell inclusion.

Experimental study on the haematogenous origin of multinucleate osteoclasts in the rat.

When the resorption zones of the distal femoral and proximal tibial epiphyseal plates of rats are examined with the electron microscope, numerous macrophages can be seen surrounding degenerated chondrocytes. Macrophages are also found in the neighbourhood of the invading sinusoids and in close proximity to multinucleate osteoclasts. Cell fusion, however, could not be observed. Repeated transfusions of female leukocytes to isohistogenic growing male rats caused an increase in the number of nuclei containing a Barr body in the osteoclasts of the males. The result suggests that leukocytes--of which only the monocytes come in question--are the source of multinucleate osteoclasts in endochondral ossification.