Amperometry in Single Cells and Tissue.

The release from cells of signaling molecules through the controlled process of exocytosis involves multiple coordinated steps and is essential for the proper control of a multitude of biological pathways across the endocrine and nervous systems. However, these events are minute both temporally and in terms of the minute amounts of neurotransmitters, hormones, growth factors, and peptides released from single vesicles during exocytosis. It is therefore difficult to measure the kinetics of single exocytosis events in real time. One noninvasive method of measuring the release of molecules from cells is carbon-fiber amperometry. In this chapter, we will describe how we undertake such measurements from both single cells and in live tissue, how the subsequent data are analyzed, and how we interpret these results in terms of their relevant physiology.

Ultrastructure of endocrine pancreatic granules during pancreatic differentiation in the grass snake, Natrix natrix L. (Lepidosauria, Serpentes).

We used transmission electron microscopy to study the pancreatic main endocrine cell types in the embryos of the grass snake Natrix natrix L. with focus on the morphology of their secretory granules. The embryonic endocrine part of the pancreas in the grass snake contains four main types of cells (A, B, D, and PP), which is similar to other vertebrates. The B granules contained a moderately electron-dense crystalline-like core that was polygonal in shape and an electron-dense outer zone. The A granules had a spherical electron-dense eccentrically located core and a moderately electron-dense outer zone. The D granules were filled with a moderately electron-dense non-homogeneous content. The PP granules had a spherical electron-dense core with an electron translucent outer zone. Within the main types of granules (A, B, D, PP), different morphological subtypes were recognized that indicated their maturity, which may be related to the different content of these granules during the process of maturation. The sequence of pancreatic endocrine cell differentiation in grass snake embryos differs from that in many vertebrates. In the grass snake embryos, the B and D cells differentiated earlier than A and PP cells. The different sequence of endocrine cell differentiation in snakes and other vertebrates has been related to phylogenetic position and nutrition during early developmental stages.

[D-cells of the gastroenteropancreatic system: development, structure, function and regeneration (history and current state of the problem)].

The present review summarizes the literature data and the results of authors' own research on the development, structure, function and regeneration of D-endocrinocytes of gastroenteropancreatic (GEP) system. The history of the research of these cells is reviewed and its current state of the problem is discussed. The information on the difference of somatostatin-producing D-endocrinocytes from other types of endocrine cells of GAP system is presented, namely, the prevalence of these cells in all the organs of the digestive system (stomach, small and large intestine, pancreas) and other systems of the body, the peculiarities of their structure and regeneration in various species of vertebrate animals and humans in embryonic development, under conditions of normal functioning and in various types of pathology. On the basis of the data on the early differentiation of D-endocrinocytes and their secretion of hormones during embryonic development, structure, cytophysiology and relationships within the general endocrinocyte population, it is suggested that D-endocrinocytes play an important role in the morpho-functional state of GEP system.

Endocrine-like cells in the chick embryo thymus express ultrastructural features of piecemeal degranulation.

The endocrine-like cells of the chick embryo thymus were studied by transmission electron microscopy and a highly characteristic pattern of cell secretion referred to as piecemeal degranulation (PMD) was identified. This is the first description of PMD in embryonic cells.

Crossreaction with an anti-Bax antibody reveals novel multi-endocrine cellular antigen.

We found a novel protein that has crossreactivity with a polyclonal anti-Bax antibody (SCBAX antibody). The protein was localized exclusively in the endocrine cells of hypothalamus, pituitary gland, and pancreatic islets. Immunohistochemical (IHC) double labeling revealed that the cells showing crossreactivity with this antibody corresponded precisely to oxytocin neurons and ACTH, alpha-MSH, and glucagon cells in rat and gerbil. By immunoelectron microscopy, the protein was localized predominantly in and just around the secretory granules in the cytoplasm but not in the mitochondria. Double-labeling IHC with the anti-Bax SCBAX antibody and two anti-Bax monoclonal antibodies (MAbs) showed that cells stained with the anti-Bax SCBAX antibody were not stained with anti-Bax MAbs except for very few cells (probably apoptotic cells). Western blotting analysis revealed that the molecular mass of the protein was approximately 55 kD, which differs from that of Bax protein (21 kD). These findings indicate that the anti-Bax SCBAX antibody recognizes not only pro-apoptotic Bax protein (a 21-kD mitochondrial protein) but also an unknown substance present in one endocrine cell group in each endocrine organ. Therefore, the protein is designated as multi-endocrine cellular antigen (MECA). MECA is probably a 55-kD protein secreted from the particular differentiated cell groups of endocrine tissues.

Fusion pore or porosome: structure and dynamics.

Electrophysiological measurements on live secretory cells almost a decade ago suggested the presence of fusion pores at the cell plasma membrane. Membrane-bound secretory vesicles were hypothesized to dock and fuse at these sites, to release their contents. Our studies using atomic force microscopy on live exocrine and neuroendocrine cells demonstrate the presence of such plasma membrane pores, revealing their morphology and dynamics at near nm resolution and in real time.

[Electron microscopic observation of endocrine cells in the antrum in response to Helicobacter pylori infection].

OBJECTIVE: To observe the ultrastructural changes of the endocrine cells in the antrum after the onset of Helicobacter pylori (Hp) infection. METHODS: Seven patients with chronic gastritis were included in this study, and toluidine blue staining and rapid urease test were performed to determine the Hp status of the gastric antral mucosa biopsies. Five patients identified as being positive for Hp constituted the test group, leaving the 2 Hp-negative patients serving as control. The endocrine cells in the antrum of the patients were sampled to observe the ultrastructural changes by transmission electron microscopy (TEM). RESULTS: TEM showed increased electron density of the secretion granules in the endocrine cells, and secretions in the parietal cells were active. CONCLUSION: The ultrastructural changes of the endocrine cells in the antrum might explain high gastrin levels after the onset of Hp infection.

Distribution of neuropeptide Y Y1 receptors in rodent peripheral tissues.

Using a sensitive immunohistochemical technique, the localization of neuropeptide Y (NPY) Y1-receptor (Y1R)-like immunoreactivity (LI) was studied in various peripheral tissues of rat. Wild-type (WT) and Y1R-knockout (KO) mice were also analyzed. Y1R-LI was found in small arteries and arterioles in many tissues, with particularly high levels in the thyroid and parathyroid glands. In the thyroid gland, Y1R-LI was seen in blood vessel walls lacking alpha-smooth muscle actin, i.e., perhaps in endothelial cells of capillaries. Larger arteries lacked detectable Y1R-LI. A distinct Y1R-immunoreactive (IR) reticulum was seen in the WT mouse spleen, but not in Y1R-KO mouse or rat. In the gastrointestinal tract, Y1R-positive neurons were observed in the myenteric plexus, and a few enteroendocrine cells were Y1R-IR. Some cells in islets of Langerhans in the pancreas were Y1R-positive, and double immunostaining showed coexistence with somatostatin in D-cells. In the urogenital tract, Y1R-LI was observed in the collecting tubule cells of the renal papillae and in some epithelial cells of the seminal vesicle. Some chromaffin cells of adrenal medulla were positive for Y1R. The problem of the specificity of the Y1R-LI is evaluated using adsorption tests as well as comparisons among rat, WT mouse, and mouse with deleted Y1R. Our findings support many earlier studies based on other methodologies, showing that Y1Rs on smooth muscle cells of blood vessels mediate NPY-induced vasoconstriction in various organs. In addition, Y1Rs in other cells in parenchymal tissues of several organs suggest nonvascular effects of NPY via the Y1R.

Ultrastructure of the rat duodenal endocrine cells after prolonged irradiation.

We propose classification of duodenal endocrine cells of intact rats based on ultrastructural signs of secretory granules and subdivided these cells into 10 basic types. The effect of long-term irradiation in a total dose of 2.5 Gy on ultrastructural organization of duodenal apudocytes was studied. Irradiation induced nonspecific changes of cell organelles in apudocytes. Differences in the ultrastructural disorganization were detected between different types of apudocyte populations and between different types of endocrine cells. Under conditions of adaptation to radiation apudocytes released the secretory product not only through molecular extrusion and exocytosis, but also via degranulation.

[Research of ultra-structural pathological changes of nervous, endocrine and immune system in heroin addicts].

OBJECTIVE: To investigate ultrastructural pathological changes of Heroin-Addicts. METHODS: Heroin-Addicts' central nervous system, endocrine system, immune system and reproductive system in 4 cases are observed by using transmission electron microscope(TEM). RESULTS: The changes of central nervous system are mitochondrion swelling, crista fragmentation and disappear. Endoplasmic reticulum dilation, nervous fibres and cell organelles reduction; mitochondrion swelling, Partial crista fragmentation and endoplasmic reticulum dilation are also found in endocrine system; Lymphocytes reduction, cytoplasm ingredient reduction and dead lymphocytes increase in immune system; in reproductive system, spermatogenic cells and cell organelles are reduced in the male and follicle disappeared in the female. CONCLUSION: Ultra-structural pathological changes of heroin-addicts are presented acute, chronic oxygen deficiency degeneration and necrosis.

Research of ultra-structural pathological changes of nervous, endocrine and immune system in heroin addicts / 法医学杂志

OBJECTIVE@#To investigate ultrastructural pathological changes of Heroin-Addicts.@*METHODS@#Heroin-Addicts' central nervous system, endocrine system, immune system and reproductive system in 4 cases are observed by using transmission electron microscope(TEM).@*RESULTS@#The changes of central nervous system are mitochondrion swelling, crista fragmentation and disappear. Endoplasmic reticulum dilation, nervous fibres and cell organelles reduction; mitochondrion swelling, Partial crista fragmentation and endoplasmic reticulum dilation are also found in endocrine system; Lymphocytes reduction, cytoplasm ingredient reduction and dead lymphocytes increase in immune system; in reproductive system, spermatogenic cells and cell organelles are reduced in the male and follicle disappeared in the female.@*CONCLUSION@#Ultra-structural pathological changes of heroin-addicts are presented acute, chronic oxygen deficiency degeneration and necrosis.

[A clinico-pathological study of neuroendocrine tumors in stomach].

OBJECTIVE: To study the pathological classification of gastric neuroendocrine tumors and its clinico-pathological significance. METHODS: Paraffin sections of totally 52 gastric neuroendocrine tumors including 42 carcinoid tumors were studied with immunohistochemical technique, which involved 9 endocrine markers of hormones antibodies and electronic microscopy for investigating the endocrine cells and the contiguous gastric mucosa of the neuroendocrine tumors. RESULTS: The 52 gastric neuroendocrine tumors were divided into three types: Type I, carcinoid, associated with atrophic gastritis, altogether 26 cases. Tumor extension was limited in the mucosa or submucosa, accompanying with hypergastrinemia and G cell hyperplasia. This type is consistently preceded by and associated with generalized proliferation of endocrine cells in the mucosa at fundus. Type II, carcinoid of sporadic type, totally 16 cases, not associating with hypergastrinemia, was more aggressive. Type III, Neuroendocrine carcinomas (10 cases), were highly aggressive tumors. CONCLUSION: A correct identification of different types of gastric endocrine tumors is important and implicit for the treatment and prognosis of neuroendocrine tumors.

Study of the effects of aging on macromolecular synthesis in various organ systems using microscopic radioautography.

In order to elucidate the effects of aging on macromolecular synthesis such as DNA, RNA, proteins, glucides and lipids in various organ systems of experimental animals and humans, systematic studies using light and electron microscopic radioautography in various organ systems including skeletal, muscular, digestive, respiratory, urinary, reproductive, endocrine, circulatory, nervous and sensory systems were studied after incorporation with macromolecular precursors. The experimental animals used were mainly ddY strain mice from embryo to postnatal days 1 and 3, weeks 1 and 2 or months 1, 2 and 6 months up to 1 and 2 years senescent stages. Animals were injected with [3H]-thymidine for DNA, [3H]-uridine for RNA, [3H]-amino acids for proteins, [3H]-glucose, [3H]-glucosamine and [35S]O4 for glucides, [3H]-glycerol for lipids and some low molecular target tracers such as hormones, inorganic substances and drugs. Results demonstrate that these precursors when incorporated into various cell types in various organs showed specific patterns of macromolecular synthesis as observed in perinatal to juvenile, mature and senescent stages. These effects of aging could answer some of the questions as to how but not why we get old.

Ultrastructure of the foregut and associated glands in the lung fluke, Paragonimus miyazakii (Digenea: Troglotrematidae), with particular reference to their functional roles.

The foregut and associated glands of a digenetic trematode, Paragonimus miyazakii, were examined in the forebody by transmission and scanning electron microscopy as well as by light microscopy, and their functional roles were discussed. The foregut is lined with a general tegument without spines and sensory receptors throughout its length, although it consists of the mouth, pharynx, and esophagus. This foregut tegument is regionally and intraregionally modified in appearance, suggesting the performance of auxiliary functions in digestion. This appearance is characterized by long, frequent cytoplasmic extensions of the apical tegument around the middle portion of the mouth and the anterior esophagus. Electron-dense granules and multimembranous and multilamellar bodies are developed in the tegument to various degrees, and elaborately in the apical layer of the prepharynx. A single type of unicellular gland is embedded in the antero-middle part of the worm in small groups. The gland cells synthesize clear secretory granules as a chief product, each granule with a pleomorphic, dense, core-like inclusion. Mature granules are elliptical in shape, approximately 500 nm in diameter, and are subsequently discharged into the prepharyngeal foregut lumen after passing through the elongated cytoplasm of the gland cell. In the prepharynx and pharynx, host blood cells are apparently processed for digestion. In the wide lumen of the esophagus, foodstuff could undergo sufficient digestion prior to absorption by the cecal epithelium.

Lipopolysaccharide-induced hyperglycemia is mediated by CHH release in crustaceans.

Septicemia in crustaceans may occur occasionally due to Gram-negative opportunistic bacteria, especially under conditions of intensive aquaculture. The lipopolysaccharide (LPS) endotoxin induces in mammals septic shock and the activation by LPS of hormone release through the hypothalamo-pituitary axis is well known. In crustaceans an increase in circulating Crustacean hyperglycemic hormone and hyperglycemia are reported to result from exposure to several environmental stressors but the metabolic and hormonal effects of LPS in vivo are undescribed. A sublethal dose of LPS (Sigma, Escherichia coli 0111:B4) was injected into at least five individuals of species representative of crustacean taxa and life habits: Squilla mantis (Stomatopoda); the Decapoda Crangon crangon and Palaemon elegans (Caridea), Nephrops norvegicus (Astacidea), Munida rugosa and Paguristes oculatus (Anomura), Pilumnus hirtellus, Macropipus vernalis, Parthenope massena, and Ilia nucleus (Brachyura). Within 3 hr an increase in blood sugar developed ranging from 26.00 +/- 8.37 sd mg/dl in M. rugosa to 201.50 +/- 95. 91 sd mg/dl in P. oculatus and a significant increase of 79% in M. rugosa up to 1300% in P. hirtellus over control levels was observed. The involvement of eyestalk hormones in this generalized response was tested on S. mantis, M. vernalis, and P. elegans; LPS injected into eyestalkless animals did not elicit a significant hyperglycemic response compared with saline-injected controls. Eyestalkless animals injected with one eyestalk equivalent homogenate in saline from untreated animals did show a change in color from red to normal likely due to red pigment concentrating hormone and a hyperglycemic response within 2 hr. Eyestalkless animals injected with homogenate from LPS-treated shrimps showed the change in color but not the hyperglycemic response. It is concluded that LPS directly, or cytokines circulated upon challenge by the endotoxin, may act on the medulla terminalis X-organ-sinus gland complex and release CHH selectively eliciting an hyperglycemic stress response, after which CHH stores become relatively depleted.