A.C. Villaro

Detection of nitric oxide synthase (NOS) in somatostatin-producing cells of human and murine stomach and pancreas.

The aim of this study was to identify by immunocytochemistry the distribution of nitric oxide synthase (NOS) in human and murine gastric epithelium. Using two different antisera specific for neuronal NOS (nNOS), we detected nNOS immunoreactivity in endocrine cells of the epithelium of the body and pyloric regions as well as in ganglion cells of the intrinsic plexi of the stomach of the three species studied. Both immunocytochemistry of contiguous sections and double immunolabeling methods showed that the nNOS-immunoreactive cells were also immunoreactive for somatostatin. Co-localization of nNOS and somatostatin has also been found in the pancreatic islets, where strong nNOS immunoreactivity appeared in scattered cells, which were peripheral in rat and mouse islets and more randomly distributed in human. The possibility of crossreactivity between the antisera against nNOS and somatostatin was ruled out by means of absorption controls. Immunocytochemical techniques were also applied to thin sections, confirming the immunostaining of gastric D-cells, which was restricted principally to the secretory granules. The possible functional implications of these findings for gastric and pancreatic physiology are discussed.

The diffuse endocrine system: from embryogenesis to carcinogenesis.

In the present review we will summarise the current knowledge about the cells comprising the Diffuse Endocrine System (DES) in mammalian organs. We will describe the morphological, histochemical and functional traits of these cells in three major systems gastrointestinal, respiratory and prostatic. We will also focus on some aspects of their ontogeny and differentiation, as well as to their relevance in carcinogenesis, especially in neuroendocrine tumors. The first chapter describes the characteristics of DES cells and some of their specific biological and biochemical traits. The second chapter deals with DES in the gastrointestinal organs, with special reference to the new data on the differentiation mechanisms that leads to the appearance of endocrine cells from an undifferentiated stem cell. The third chapter is devoted to DES of the respiratory system and some aspects of its biological role, both, during development and adulthood. Neuroendocrine hyperplasia and neuroendocrine lung tumors are also addressed. Finally, the last chapter deals with the prostatic DES, discussing its probable functional role and its relevance in hormone-resistant prostatic carcinomas.

Neuronal nitric oxide synthase immunoreactivity in the respiratory tract of the frog, Rana temporaria

SummaryPhysiological and histochemical studies have recently supported the notion that nitric oxide (NO) is the transduction signal responsible for the non-adrenergic, non-cholinergic relaxation of the vasculature as well as the airways of the mammalian lung. We report the presence of immunoreactivity to NO synthase (NOS) in nerve cell bodies and nerve fibres in the neural plexus of the buccal cavity and lungs of the frog, Rana temporaria, using the indirect immunocytochemical technique of avidin-biotin and the NADPH-diaphorase technique. The neural ganglia located next to the muscle layer and within the connective tissue of the buccal cavity were partially immunoreactive for NOS. In the lungs, NOS immunoreactivity occurred in nerve cell bodies, as well as in both myelinated and unmyelinated nerve fibres. Fine nerve fibres immunoreactive to NOS were observed within the muscle fibre bundles and next to the respiratory epithelium. Both the presence of NOS immunoreactivity and the positive histochemical reaction for NADPH-diaphorase in the neural plexus of amphibian respiratory tract suggests a broad evolutionary role for NO as a peripheral neurotransmitter.

An immunocytochemical and ultrastructural study of the larval anterior intestine of the frog Rana temporaria, with especial reference to endocrine cells.

Endocrine cells of the larval intestine of Rana temporaria tadpoles have been identified by argyrophilic, immunocytochemical and electron-microscopical techniques. Scarce endocrine cells have been found in both the short non-absorptive zone immediately following the stomach, and in the rest of the anterior intestine. Endocrine cells are frequently seen to extend a cytoplasmic process towards the lumen. Immunoreactivity for serotonin, somatostatin, bombesin and cholecystokinin-8 has been detected. According to the ultrastructural traits of the endocrine granules, three larval intestinal endocrine populations have been differentiated.

Malpighian tubules of Formica polyctena (Hymenoptera): Light and electron microscopic study

The study of semithin (1 μ in thickness) and thin sections of Epon‐embedded material reveals that Malpighian tubules of Formica polyctena are composed of a single cellular type throughout their length. No differentiated regions were observed. Ultrastructural traits of the epithelial cells are those of highly metabolic cells engaged in water and ion transport: large euchromatic nucleus, several nucleoli, abundant mitochondria, a conspicuous brush border, and basolateral infoldings. Lysosomes, smooth endoplasmic reticulum, and intercellular junctions are also well developed. Mineral concretions occur within the cells and in the lumen. However, the degree of development of these cytological traits is variable among the different cells. The tubules drain into the gut through a specialized posterior region of the midgut or ampulla. In this region, the epithelial cells, although usually taller and showing conspicuous basal infoldings, display cytological structures similar to those of the Malpighian tubules.

Light and electron microscopic study of the hindgut of the ant (Formica nigricans, Hymenoptera): II. structure of the rectum

The study of the ileum of the ant Formica nigricans by light and electron microscopy revealed the existence of three differentiated regions: proximal, middle, and distal ileum. The middle region constitutes most of the length of the organ. Its wall is made up by a folded simple epithelium lined by a cuticle, which is surrounded by an inner circular muscle layer and various external longitudinal muscle fibers adjacent to the hemolymph. A subepithelial space is present between the epithelium and the circular muscle layer. Epithelial cells show extensive infoldings of the apical, and to a lesser extent the basolateral plasma membrane. Apical infoldings are characterized by the presence of 10‐nm particles (portasomes) covering the cytoplasmic side of the membrane. Mitochondria are abundant throughout the cytoplasm, although they mainly are present underneath the apical infoldings. Lateral borders of epithelial cells display an apical junctional complex, mainly constituted by a long and convoluted pleated septate junction. These features support the view that epithelial cells in the middle ileum are specialized in ion solutes and water transport. The proximal ileum connects with the ampulla into which the Malpighian tubules drain. As opposed to the middle ileum, epithelial cells of the proximal ileum show less developed basolateral infoldings, and the apical plasma membrane is devoid of portasomes and only occasionally invaginates. These features suggest that the proximal ileum plays no relevant role in ion and water transport. The distal ileum penetrates into the rectal sac, forming a valve‐like structure; this region presumably controls the amount of urine reaching the rectum. J. Morphol. 242:189–204, 1999.

Structural study of the frog Rana temporaria larval stomach

The gastric wall of Rana temporaria tadpoles consists of a well-developed mucosa and thin muscular and serosa layers. Three cellular types--mucous, ciliated and endocrine cells--make up the lining epithelium. Different types of endocrine cells exist. Argyrophylic endocrine cells can be recognized in semithin sections of plastic-embedded material while non-argyrophylic endocrine cells can only be identified under the electron microscope. Glands are composed mainly of well-differentiated oxyntic cells and, occasionally, scarce endocrine cells. Oxyntic cells show abundant mitochondria and smooth endoplasmic reticulum, but do not contain zymogen granules as do those present in adults. Secretory canaliculi with microvilli are also well-developed. The lamina propria contains numerous vascular sinuses and nerve bundles which innervate the endothelium and some endocrine cells. The neuroendocrine regulation of frog gastric functions seems therefore to have developed in young tadpoles. Nerve fibers also innervate the muscular propria, which is composed of a single layer of smooth muscle cells. Underlying the muscle, connective fibers and a flattened layer of mesothelial cells make up the serosa. In summary, the structure of the frog larval stomach shows a well-differentiated histological pattern, especially referring to surface epithelium and glands. Some of the histological traits will also be present in adult frogs while others are characteristic of the tadpoles stage.

Gut endocrine cells in the snail Helix aspersa

A microscopic study of the endocrine cells present in the gut of the snail Helix aspersa is made. Electron microscopy is necessary in most cases to identify the enteroendocrine cells, since neither silver impregnations nor immunocytochemical staining have rendered positive results. Endocrine cells are scarce and rest on the basement membrane. They display a clear cytoplasm and variable amounts of small (143 nm) secretory granules of diverse electron-density. They are ovoid or rounded and possess apical processes which extend into the lumen of the gut. The nucleus, located in the basal region of the cell, presents characteristic cytoplasmic indentations. Intraepithelial nerve bundles in contact with endocrine cells are present.

Metamorphic changes in the stomach of the frog Rana temporaria tadpoles

The histological transformation of amphibian stomach during metamorphosis was studied in the frog Rana temporaria. The earliest metamorphic changes occur shortly before regression of the cloacal piece of tail and appearance of forelegs. Autolysis of primary, larval epithelial cells and activity of phagocytes lead to regression of the apical mucosa, which is shed into the gastric lumen. Histogenesis takes place from the very beginning of metamorphosis in the basal region of the mucosa; undifferentiated, regenerative cells, arranged in small compact cords and surrounded by a thick basement membrane, give rise to secondary lining epithelium and glands. Lining epithelial cells differentiate into a typical mucosecretory epithelium. Oxyntic cells present in larval glands are substituted by both ion- and protein-secreting oxyntic-peptic cells. During metamorphosis, connective and muscular tissues markedly increase, a submucosa, not present in larval tadpoles, gradually develops. A muscularis mucosae is also formed and the muscular propria becomes thicker. At late metamorphosis, folded structures involving both mucosa and submucosa develop, increasing the luminal surface as in adults. Removal of the larval gastric mucosa and its replacement by a new, adult-type definitive one, together with development of peripheral connective and muscular tissues, account for metamorphosis of tadpole stomach.

Immunocytochemical localization of a vacuolar-type ATPase in Malpighian tubules of the ant Formica polyctena

The presence of a vacuolar-type ATPase in Malpighian tubules of the ant Formica polyctena was investigated immunocytochemically, using antibodies to vacuolar ATPases of Manduca sexta midgut and bovine kidney. Specific labelling was observed at the brush border of the epithelium extending along the entire length of the tubules. These findings agree with the current view that a vacuolar ATPase is situated at the apical membrane of Malpighian tubule cells and other insect epithelial cells, being the energizing element of an electrogenic potassium pump. When antibodies were tested on tubules in different secretion conditions prior to fixation, no differences were observed in the distribution of the vacuolar ATPase.