Yohichi Satoh

Immunohistochemical and histochemical evidence for the presence of noradrenaline, serotonin and gamma-aminobutyric acid in chief cells of the mouse carotid body

The immunohistochemical study revealed tyrosine hydroxylase (TH), dopamine β-hydroxylase (DBH), phenylethanolamine N-methyltransferase (PNMT), serotonin, glutamate decarboxylase (GAD) and γ-aminobutyric acid (GABA) immunoreactivities in the mouse carotid body. TH and DBH immunoreactivities were found in almost all chief cells and a few ganglion cells, and in relatively numerous varicose nerve fibers of the carotid body. The histofluorescence microscopy showed catecholamine fluorescence in almost all chief cells. However, no PNMT immunoreactivity was observed in the carotid body. Serotonin, GAD and GABA immunoreactivities were also seen in almost all chief cells of the carotid body. From combined immunohistochemistry and fluorescence histochemistry, catecholamine and serotonin or catecholamine and GABA were colocalized in almost all chief cells. Thus, these findings suggest that noradrenaline, serotonin and GABA may be synthesized and co-exist in almost all chief cells of the mouse carotid body and may play roles in chemoreceptive functions.

Ganglion cells immunoreactive for catecholamine-synthesizing enzymes, neuropeptide Y and vasoactive intestinal polypeptide in the rat adrenal gland

Immunohistochemistry has been used to demonstrate tyrosine hydroxylase (TH), dopamine-β-hydroxylase (DBH), phenylethanolamine N-methyltransferase (PNMT), neuropeptide Y (NPY) and vasoactive intestinal polypeptide (VIP) immunoreactivities, and acetylcholinesterase (AChE) activity was demonstrated in rat adrenal glands. The TH, DBH, NPY and VIP immunoreactivities and AChE activity were observed in both the large ganglion cells and the small chromaffin cells whereas PNMT immunoreactivity was found only in chromaffin cells, and not in ganglion cells. Most intraadrenal ganglion cells showed NPY immunoreactivity and a few were VIP immunoreactive. Numerous NPY-immunoreactive ganglion cells were also immunoreactive for TH and DBH; these cells were localized as single cells or groups of several cells in the adrenal cortex and medulla. Use of serial sections, or double and triple staining techniques, showed that all TH- and DBH-immunoreactive ganglion cells also showed NPY immunoreactivity, whereas some NPY-immunoreactive ganglion cells were TH and DBH immunonegative. NPY-immunoreactive ganglion cells showed no VIP immunoreactivity. AChE activity was seen in VIP-immunopositive and VIP-immunonegative ganglion cells. These results suggest that ganglion cells containing noradrenaline and NPY, or NPY only, or VIP and acetylcholine occur in the rat adrenal gland; they may project within the adrenal gland or to other target organs. TH, DBH, NPY, and VIP were colocalized in numerous immunoreactive nerve fibres, which were distributed in the superficial adrenal cortex, while TH-, DBH- and NPY-immunoreactive ganglion cells and nerve fibres were different from VIP-immunoreactive ganglion cells and nerve fibres in the medulla. This suggests that the immunoreactive nerve fibres in the superficial cortex may be mainly extrinsic in origin and may be different from those in the medulla.

Effect of live and heat-killed bacteria on the secretory activity of Paneth cells in germ-free mice

SummaryGerm-free mice were given live or heat-killed facultative anaerobes, and the ultrastructure of ileal Paneth cells was quantitatively examined with special reference to secretory granules showing a bipartite substructure (central core and peripheral halo). After administering live or heatkilled bacteria, there was a decrease in the area occupied by the cores of secretory granules in Paneth cells, and exocytosed core material was observed in the crypt lumen. There were no changes in the area occupied by the halo of secretory granules. None of the examined Paneth cells phagocytosed bacteria. It is concluded that certain bacteria may affect the secretion of antibacterial agents contained in the secretory granules of Paneth cells.

Quantitative Light Microscopic Observations on Paneth Cells of Germ-Free and Ex-Germ-Free Wistar Rats

Germ-free rats were inoculated with bacteria from feces of SPF rats, and the Paneth cells in the ileal crypts were observed at different time intervals after inoculation. 12 h after inoculation, the Paneth cells showed a striking degranulation and the occurrence of supranuclear vacuoles. The Paneth cell area was significantly reduced. Four days to 3 weeks after inoculation, the secretory granules of Paneth cells were abundant, and both number and area of Paneth cells showed a progressive increase coming close to the data in SPF rats. The present study demonstrates interrelationships between secretory activity of Paneth cells and the microbial milieu in the small intestine and that the presently used experimental model is well suited for examining the histophysiology of Paneth cells. Moreover, the results suggest that Paneth cell numbers may also be closely related to crypt length.

Quantitative electron microscopic observations on Paneth cells of germfree and ex-germfree Wistar rats.

SummaryUltrastructural changes of Paneth cells of germfree (Gf) rats which had been inoculated with bacteria-containing feces from conventionally-reared (SPF) rats were quantitatively examined. 12 and 24 h after inoculation, the Paneth cells showed a striking decrease in the number of secretory granules and the occurrence of large vacuoles. Phagosomes containing bacteria were not seen. After 4 days, the secretory granules reaccumulated and smooth-surfaced apical vesicles increased in number. It is discussed that the large vacuoles may be related to membrane-retrieval events following the massive extrusion of secretory granules whereas the apical vesicles appear to serve this function when exocytosis is not pronounced. In addition to the large secretory granules ca. 10% of Paneth cell profiles contained a few dense-cored vesicles measuring about 150 nm in diameter which resemble peptidergic neurosecretory granules.

Configuration of myoepithelial cells in various exocrine glands of guinea pigs

To study the configuration of myoepithelial cells, we isolated glandular endpieces of various guinea pig glands by collagenase, and visualized the myoepithelial cells by immunohistochemistry for actin, or by Bodipy-phallacidin, under both a light microscope and laser scanning confocal microscopes. In parotid and mandibular glands, the glandular acini were small (about 20–30 μm diameter) and spherical, and each acinus had one or two myoepithelial cells attached that were stellate in shape (central cell body and four to six thin processes). Most of the basal surface of the glandular cells was not covered by myoepithelial cells, and processes often extended to the neighboring acinus. The tubular glandular endpieces of the major sublingual gland, which secretes a mucous substance, were almost fully encircled by bandlike myoepithelial cells (about 3–6 μm wide). Although there were many differences between the lacrimal gland and the Harderian gland (e.g., the secretory product of the lacrimal gland was mucous, and glandular lumina were narrow; the Harderian gland secreted lipids and showed wide lumina), the outer contours of both glandular endpieces were the same (about 50–100 μm diameter, ellipsoid or spherical in shape). In both glands, 5–20 stellate myoepithelial cells were attached onto a glandular endpiece, and their arrangement had a lacy appearance. Actin filaments in myoepithelial cells aggregated and formed bundles in the broad processes and cell bodies. The bundles ran across the cell body, but there was no point where the bundles converged. In the arborization, some distal processes reversed their direction. We conclude that the configuration of myoepithelial cells depends on the outer contour of the glandular endpieces rather than on the secretory material or luminal width. The variety of myoepithelial cell configurations in the different exocrine glands we examined suggests that it is quite difficult to assign to myoepithelial cells the general role of expelling secretory products from glandular lumina. These cells seem to maintain the contour of the glandular endpieces, serving as the exoskeleton of the endpieces.

Atropine inhibits the degranulation of Paneth cells in ex-germ-free mice

SummaryPrevious studies have shown that the secretory products of Paneth cells contain antibacterial agents (lysozyme, IgA) that are affected by the bacterial milieu in the intestine. To investigate whether Paneth-cell secretion is controlled via cholinergic mechanisms, the ultrastructure of Paneth cells was studied in four animal groups: (1) germfree (GF) control mice (Jcl: ICR [GN], male, 13 weeks old), (2) GF mice injected subcutaneously with atropine sulfate (200 mg/kg body weight, dissolved in physiological saline 20 mg/ml), (3) ex-GF mice inoculated with feces from specific-pathogen-free (SPF) mice, and (4) ex-GF mice injected with atropine and inoculated with feces from SPF mice. In ex-GF mice inoculated with feces, 70–90% of the Paneth cells showed fewer secretory granules than those from GF mice (p<0.01). Approximately 30% of the Paneth cells had a large vacuole (3–10 μm diameter) in the apical cytoplasm. Exocytosed electron-dense material from secretory granules was observed in a few crypt lumens. In ex-GF mice inoculated with feces and given atropine, about 90% of the Paneth cells contained numerous secretory granules, like those in GF control mice, but vacuolated Paneth cells and exocytotic figures were rare; thus the secretion of Paneth cells was blocked by atropine. It is therefore possible that the bacterial milieu in the intestine affects the secretory activity of Paneth cells via cholinergic mechanisms.

Immunohistochemical observations of immunoglobulin A in the Paneth cells of germ-free and formerly-germ-free rats.

SummaryThe localization of secretory immunoglobulin A (IgA) in Paneth cells was immunohistochemically studied in germ-free (Gf) and ex-Gf rats that had been injected with feces obtained from specific-pathogen-free (SPF) rats. In Gf as well as SPF rats, the secretory granules of Paneth cells and the brush borders of crypt cells exhibited IgA immunoreactivity. At 12 and 24 h after inoculation, it was found that, concomitant with the occurrence of considerable degranulation, the IgA immunoreactivity in Paneth cells disappeared, except of the margin of supranuclear vacuoles. In contrast, the IgA immunoreactivity of the crypt-cell brush borders was unchanged. Four days after inoculation, secretory granules exhibiting IgA immunoreactivity reaccumulated in Paneth cells. The present study suggests that Paneth cells regulate the bacterial milieu in the intestine by releasing secretory granules containing IgA into the crypt lumen.

Effect of carbamylcholine on Harderian gland morphology in rats

SummaryTo determine the effect of cholinergic secretagogue on the Harderian gland of rats, several light- and electron-microscopic parameters were morphometrically assessed at different time intervals after carbamylcholine injection. In controls, two types of glandular cells (type A cells having 40–55 large vacuoles per cell profile and type B cells containing 30–38 smaller vacuoles per cell profile) and myoepithelial cells were recognized. At 5 min after injection of carbamylcholine, when rats secreted “bloody tears”, many alveoli showing narrower lumina and exocytotic figures in both types of cells were observed. Some vacuoles, which were covered by thin cytoplasmic sheets, protruded into the alveolar lumina. However, there was no evidence of apocrine or holocrine secretion. At 30 min and 120 min after injection, most of the alveolar lumina were dilated, and a pronounced decrease in the number of vacuoles in the glandular cells was observed. At 300 min after injection, the secretory vacuoles in both cell types reaccumulated. Transitional forms between the two cell types were not observed. The two types of Harderian gland cells can therefore be considered independent populations rather than different secretory stages of the same cell. It appears that the secretory process of the Harderian gland of rat is affected by cholinergic stimulation of the two types of glandular cells and of myoepithelial cells.

Substance P immunoreactivity in rat von Ebner's gland.

SummaryThe present immunohistochemical study revealed substance P-immunoreactive neuronal elements in the von Ebners gland of rats. Immunoreactive ganglion cells were observed as single cells or groups of several immunoreactive ganglion cells among intra-lingual muscles, at the base of the vallate papillae and near the von Ebners gland. Very numerous substance P-immunoreactive varicose nerve fibres ran closely associated with the serous cells and excretory duct cells, and were seen to run along blood vessels in the gland. Since substance P-immunoreactive ganglion cells were present near the glands, the immunoreactive varicose nerve fibres in the von Ebners gland may be partly derived from the intra-lingual ganglion cells. These substance P-immunoreactive varicose nerve fibres may have an effect on the secretory activity of the serous cells and duct cells, and on the vasodilation of blood vessels of the von Ebners gland. Actin immunoreactivity was seen in numerous myoepithelial cells embracing serous cells and duct cells, and in the smooth muscle cells of blood vessels of the gland. By using a double immunolabelling technique with anti-substance P and anti-actin sera, substance P-immunoreactive varicose nerve fibres were found to be in close contact with myoepithelial cells.