Immunoelectron microscopic study for histamine in the gastric enterochromaffin-like cells of rats treated with the proton pump inhibitor lansoprazole

The enterochromaffin-like (ECL) cells of the gastric mucosa in animals play an important role in gastric acid secretion. They contain few granules and numerous secretory vesicles and microvesicles. They operate under the control of circulating gastrin. In the present study, we conducted an immunoelectron microscopic study for histamine (HA) in the ECL cells of rats given the proton pump inhibitor lansoprazole (LP), which is known to induce hypergastrinemia. The pre-embedding indirect immunoperoxidase procedure utilized a mouse monoclonal antibody AHA-2 against glutaraldehyde-conjugated HA. Rats received LP (50 g/kg per day, subcutaneously) over a period of a month, and developed hypertrophy of the ECL cells in the stomach. It was clearly demonstrated that HA was located to a much higher degree in the cytoplasm of ECL cells of LP-treated rats than in normal rats. HA immunoreactivity was observed in the cores of the granules and secretory vesicles of the ECL cells in all the rats, but in the LP-treated rats it was observed in the cores of the newly developed vacuoles as well. These results may suggest that HA may be actively generated in the cytoplasm of the hypertrophic ECL cells of LP-treated rats. Also suggested in the present study is that HA is instrumental in the transformation of granules into secretory vesicles and in their consequent enlargement, and that vacuoles are formed by the fusion of large secretory vesicles. Furthermore, the finding that relatively little HA immunoreactivity existed in the vacuoles may suggest that the vacuoles actively degrade superfluous secretory products (for example, HA) through enhanced autophagocytosis and/or oxidative stress. Another possibility may be that the membrane-bounded structure regarded as the vacuoles in this study might actually be an invagination structure produced as a result of successive series of exocytosis through which the secretory vesicles actively and rapidly release HA.     

Heterogeneity of dendritic cells in rat apical periodontitis

Dendritic cells are important for the induction of T-lymphocyte-mediated immunity by acting as antigen-presenting cells. We have previously reported that dendritic cells are prevalent in the chronic non-expanding phase of rat apical periodontitis. To characterize these cells further, immunoelectron microscopy with three dendritic cell markers (CD11c, OX6, OX62) was conducted for samples from rat models of apical periodontitis. Dendritic cells were divided into two types (type I or type II). Most of the type I dendritic cells expressed CD11c, showed an irregular large profile, had typical cytoplasmic processes, and were recognized as the major dendritic cell population. Most of the type II dendritic cells expressed OX62, showed oval small profiles with a few thin short processes, and were sometimes observed infiltrating from blood vessels. Cell-to-cell contacts between type I dendritic cells and lymphocytes were the most frequently observed associations. These results suggest that dendritic cells are composed of heterogeneous populations that exhibit different phenotypes, morphologies, and maturation/differentiation/activation. This study was supported by Grants-in-Aid for Scientific Research (no. 11470402 to T.O., and nos. 15791091 and 18791393 to T.K.) from the Japan Society for the Promotion of Sciences.     

Transport of membrane-bound macromolecules by M cells in follicle-associated epithelium of rabbit Peyer's patch

Summary M cells in Peyer's patch epithelium conduct transepithelial transport of luminal antigens to cells of the mucosal immune system. To determine the distribution of specific lectin-binding sites on luminal membranes of living M cells and to follow the transport route of membranebound molecules, lectin-ferritin conjugates and cationized ferritin were applied to rabbit Peyer's patch mucosa in vivo and in vitro. The degree to which binding enhances transport was estimated by comparing quantitatively the transport of an adherent probe, wheat germ agglutinin-ferritin, to that of a nonadherent BSA-colloidal gold probe. When applied to fixed tissue, the lectins tested bound equally well to M cells and columnar absorptive cells. On living mucosa, however, ferritin conjugates of wheat germ agglutinin and Ricinus communis agglutinins I and II bound more avidly to M cells. Absorptive cells conducted little uptake and no detectable transepithelial transport. Lectins on M cell membranes were endocytosed from coated pits, rapidly transported in a complex system of tubulocisternae and vesicles, and remained adherent to M cell basolateral membranes. Cationized ferritin adhered to anionic sites and was similarly transported, but was released as free clusters at M cell basolateral surfaces. When applied simultaneously to Peyer's patch mucosa, wheat germ agglutinin-ferritin was transported about 50 times more efficiently than was bovine serum albumin-colloidal gold.     

Postnatal changes in the ultrastructure of the rat olfactory epithelium: the supranuclear region of the supporting cells

Summary The present electron-microscopical study reports ultrastructural changes occurring in the supranuclear region of the supporting cells of the rat olfactory epithelium during the first 16 days of postnatal life. These changes are concerned with the enclosure of receptor cell dendrites and an increase in the amount of smooth endoplasmic reticulum, which has a specific distribution in the supporting cell. An increase in microvillous projections at the free cell surface is also observed. Moreover, this report demonstrates that the cytoarchitecture of the apical portion of the olfactory epithelium at the 16th day of postnatal life is similar, with respect to the relationship between supporting cells and receptor cell dendrites, to that of adult animals.     

Interaction of Listeria monocytogenes with the intestinal epithelium

Listeria monocytogenes is a food-borne pathogen that must cross the intestinal epithelial barrier to reach its target organs. We have investigated the importance of M cells in translocation using an experimental mouse model and a novel, recently described in vitro coculture system that mimics the follicle-associated epithelium (FAE). Our data demonstrate that L. monocytogenes does not require, nor specifically use, M cells of the FAE to cross the gut. We also show that bacterial translocation is rapid and L. monocytogenes can attach very efficiently to exposed basal lamina of the small intestine indicating an important role for extracellular matrix proteins.     

Morphological alteration of gut-associated lymphoid tissue after long-term total parenteral nutrition in rats

Summary The morphological alteration of gut-associated lymphoid tissue (GALT) induced by long-term absence of dietary stimulation was investigated. Male Wistar rats weighing 230 g were maintained with total parenteral nutrition (TPN). Control rats were allowed to have the same amount of the solution orally. After two weeks, the morphological alteration of GALT was examined. Although no significant difference in weight gain was noted between the two groups, the area comprised by Peyer's patches was decreased in TPN rats. The number of transported lymphocytes and the ratio of helper T (T_h) cells to suppressor/cytotoxic T (T_s/c) cells in intestinal lymph were lowered after TPN treatment. In an immunohistochemical study of the rat ileum, the number of T cells and the T_h/T_s/c ratio were decreased both in the intraepithelial spaces and in the lamina propria of TPN rats. The percentage of interleukin-2 receptor-positive cells and the number of IgA-containing cells in the lamina propria were significantly reduced in TPN rats. These results suggest that dietary stimulation might play a role in the maintenance of GALT function and morphology.     

Rostro-caudal patterning of receptor-expressing olfactory neurones in the rat nasal cavity

The rostro-caudal extent of odorant receptor expression zones in the rat olfactory epithelium was analysed by means of in situ hybridization. Three broad non-overlapping zones were identified that extended along almost the entire anterior-posterior axis; each zone was composed of several separate bands running anterior to posterior throughout the olfactory epithelium. Superimposed onto these broad zones was the expression area of a particular receptor subtype (OR37); it was restricted to a small region of the epithelial sheet with a high density of reactive neurones in the centre and declining numbers towards the periphery of the region. A quantitative evaluation of the reactive cells revealed that, despite their diferent distribution patterns, all receptor subtypes were expressed in an equal number of neurones.     

A scanning electron-microscopic study of the peripolar cell of the rat renal glomerulus

Summary The interior of Bowman's capsules of rat kidneys has been examined by scanning electron microscopy, and a distinctive population of cells around the exposed vascular poles of glomerular tufts were identified. The cells were situated in the annular groove at the root of the glomerulus, between the parietal epithelial cells and the podocytes. These peripolar cells were dendritic cells with long processes embracing the glomerular arterioles. Up to three peripolar cells were present at each vascular pole and they were mainly distributed in the glomeruli of the outer third of the renal cortex. This first detailed study of the surface morphology of the glomerular peripolar cell supports the suggestion that changes in the diameter of the polar region of the glomerular tuft may cause variations in stretching of the cuff of peripolar cells, and hence modulation of their secretory activity.     

Stage-specific localization of cytoskeletal actin mRNA in murine seminiferous tubules and intestinal epithelia as demonstrated by in-situ hybridization

Summary In-situ hybridization experiments have been performed using isoactin ( and )-specific riboprobes in various tissues of the rat and mouse. Distribution of the grains of actin mRNAs for both and types was similar throughout sections of the rat testis. Although both mRNAs were evenly distributed in the seminiferous tubule, extremely heavy labeling was observed in about 10% of the seminiferous tubules that could be identified as stage XII of spermatogenesis. At high magnification, grains of the mRNA were found in the cytoplasm of elongating spermatids and in the Sertoli cell cytoplasm at the adluminal side. Much higher density of the grains of mRNA was observed in the neck region of the spermatids at stage XII. Thus, the dense distribution of cytoskeletal actin mRNAs is stage-specific in the tubule during spermatogenesis in the rat. The high expression of both and actin mRNAs was also observed in the epithelial cells of the intestinal crypts.     

Expression of GABA transporters,GAT-1 and GAT-3, in the cerebral cortex and thalamus of the rat during postnatal development

The cellular and subcellular localization of two GABA transporters, GAT-1 and GAT-3, was investigated using immunocytochemical methods in the rat cerebral cortex and thalamus during postnatal development. The distribution of the transporters is compared with that of the neuronal marker GABA, and with that of vimentin and of glial fibrillary acidic protein, which identify immature and mature astrocytes, respectively. Our observations show that the two transporters are already expressed at birth in both brain areas with the same cellular localization as in adult rats, as GAT-1 is present in growth cones and terminals only in the cortex, whereas both transporters are expressed in astrocytes in the cortex and thalamus. The distribution of GAT-1 and GAT-3 undergoes postnatal changes reflecting in general the neurogenetic events of the neocortex and thalamus and, more specifically, the maturation of GABAergic innervation. The adult-like pattern of expression is achieved in the third postnatal week in the cortex and in the second postnatal week in the thalamus. The early expression of GAT-1 in GABAergic terminals confirms previous studies showing the existence of neuronal mechanisms of GABA uptake from the embryonic stages. As for the glial localization, the precocious existence of two astrocytic GABA transporters suggests that they operate through different functional mechanisms from birth, whereas their exclusively glial expression in the thalamus indicates that the astroglia plays a major role in the transport, recycling and metabolism of thalamic GABA.     

Generation of monoclonal antibodies detecting specific epitopes in olfactory and respiratory epithelia

Summary Two panels of monoclonal antibodies have been generated, each panel having a distinct specificity for antigens located in the ciliary zone of either the olfactory or respiratory epithelium of rats. Tissue specificity was confirmed in enzyme-linked immunosorbent assays on membrane fractions from various tissues. During ontogeny, the expression of olfactory-specific antigens preceeds that of respiratory-specific antigens; this observation correlates with differences in the genesis of the respective cilia type and confirms that different molecular entities are recognized. A spatial segregation of immunoreactivity in the chemosensory epithelium was observed for one of the olfactory-specific monoclonal antibodies; negative zones were located in the dorsal recess of the nasal cavity and on the tips of the turbinates. Olfactory-specific antibodies reacted with distinct polypeptide bands on Western blots from olfactory ciliary preparations.     

Evidence that an extrahypothalamic pituitary corticotropin-releasing hormone (CRH)/adrenocorticotropin (ACTH) system controls adrenal growth and secretion in rats

Within two weeks, hypophysectomy induced in rats a striking decrease in the level of circulating ACTH (the concentration of which was at the limit of sensitivity of our assay system), coupled with a net reduction in the plasma corticosterone concentration and an evident adrenal atrophy. Zona fasciculata, the main producer of glucocorticoids, was decreased in volume, due to a lowering in both the number and average volume of its parenchymal cells. Subcutaneous ACTH infusion (0.1 pmol·min^-1), administered during the last week following hypophysectomy, restored the normal blood level of ACTH and completely reversed all effects of hypophysectomy on the adrenals. Subcutaneous infusion for one week with -helical-CRH or corticotropin-inhibiting peptide (1 nmol·min^-1), which are competitive inhibitors of CRH and ACTH, evoked a further significant lowering of plasma corticosterone concentration and markedly enhanced adrenal atrophy in hypophysectomized rats. These findings strongly suggest that an extrahypothalamic pituitary CRH/ACTH system may be involved in the maintenance of the growth and steroidogenic secretory activity of the rat adrenal cortex.     

The three-dimensional cytoarchitecture of the interstitial tissue in the rat kidney

Summary The cytoarchitecture of the interstitial tissue of the rat kidney was studied by combined scanning and transmission electron microscopy. The renal interstitium is composed of an elaborate network of stellate sustentacular cells. In the cortex, sustentacular cells radiate thin branching processes to form a fine reticulum, which supports intertubular spaces. In the medulla, these cells extend thick processes horizontally along the basal surfaces of the thin limbs or vasa recta, reinforcing their attenuate walls. The horizontal processes connect with each other at their terminals, compartmentalizing the interstitial space into thin layers. The medullary sustentacular cells contain abundant small lipid droplets. The network of sustentacular cells houses vasa recta, keeping them in parallel position to each other and to the tubules. The arterial vasa recta are accompanied by pericytes, which frequently contain lipid droplets larger in size than those in the sustentacular cells. Venous vasa recta extend numerous basal microvilli, which anchor the venous wall to adjacent tubules or vessels. Numerous free cells, round in shape, are found in the sustentacular cell network, especially in the cortex. They consist of macrophages and occasional lymphocytes. Some macrophages extend long pseudopodia, while others make intimate contact with lymphocytes, suggesting their high level of activity.     

The effects of ageing on the morphology and function of the zonae fasciculata and reticularis of the rat adrenal cortex

Summary The morphological counterpart of the well-known age-dependent marked impairment of glucocorticoid secretion of rat adrenals was investigated by use of morphometric techniques. For this purpose 4-, 8-, 16- and 24-month-old rats were studied. Despite the notable lowering of both basal and ACTH-stimulated production of corticosterone by collagenase-dispersed inner adrenocortical cells, ACTH and corticosterone plasma concentrations displayed significant increases with ageing. Zona fasciculata (ZF) and zona reticularis (ZR) showed a notable hypertrophy in aged rats, which was due to rises in both the average volume and number of their parenchymal cells. The hypertrophy of ZF and ZR cells was in turn associated with increase in the volume of the mitochondrial compartment and proliferation of smooth endoplasmic reticulum, i.e., the two organelles involved in steroid-hormone synthesis. All these morphologic changes, conceivably due to the chronic exposure to high levels of circulating ACTH, are interpreted as a response enabling ZF and ZR to compensate for their age-dependent lowering in glucocorticoid secretion. Stereology also demonstrated that ZF and ZR cells underwent a striking age-related lipid-droplet repletion. Lipid droplets are the intracellular stores of cholesterol esters, the obligate precursors of steroid hormones in rats. This finding is in keeping with the contention that the mechanism underlying the age-dependent decline in rat-adrenal glucocorticoid secretion mainly involves impairments of the utilization of intracellular cholesterol previous to its intramitochondrial transformation to pregnenolone.     

Anatomical mapping of choline acetyltransferase (ChAT)-like and glutamate decarboxylase (GAD)-like immunoreactivity in outer hair cell efferents in adult rats

Summary The distribution of choline acetyltransferase (ChAT)-like and glutamate decarboxylase (GAD)-like immunoreactivity in the cochleae of 15 adult Wistar white rats was investigated using the peroxidase-antiperoxidase (PAP) technique. A monoclonal antibody to ChAT and a polyclonal antiserum to GAD were used. Immunoreaction was investigated quantitatively, in the electron microscope, on tangential sections of the tunnel of Corti and the rows of outer hair cells. ChAT-like and GAD-like immunoreactivity was found in all efferent nerve fibres in the tunnel of Corti and in all efferent synapses on the outer hair cells. A coexistence of ChAT and GAD in the efferent system to the outer hair cells of the rat is therefore assumed.     

Fine structural study of interstitial cells associated with the deep muscular plexus of the rat small intestine,with special reference to the intestinal pacemaker cells

Two types of interstitial cells have been demonstrated in close association in the deep muscular plexus of rat small intestine, by electron microscopy. Cells of the first type are characterized by a fibroblastic ultrastructure, i.e. a well-developed granular endoplasmic reticulum, Golgi apparatus and absence of the basal lamina. They form a few small gap junctions with the circular muscle cells and show close contact with axon terminals containing many synaptic vesicles. They may play a role in conducting electrical signals in the muscle tissue. Cells of the second type are characterized by many large gap junctions that interconnect with each other and with the circular muscle cells. Their cytoplasm is rich in cell organells, including mitochondria, granular endoplasmic reticulum and Golgi apparatus. They show some resemblance to the smooth muscle cells and have an incomplete basal lamina, caveolae and subsurface cisterns. However, they do not contain an organized contractile apparatus, although many intermediate filaments are present in their processes. They also show close contacts with axon terminals containing synaptic vesicles. These gap-junction-rich cells may be regular components of the intestinal tract and may be involved in the pacemaking activity of intestinal movement.