Distribution of F-actin and the membrane skeletal proteins fodrin and ankyrin in gastric parietal cells of the rat was examined by immunofluorescence and immunoelectron microscopic techniques of frozen sectiens. Rats were treated with gastrin or famotidine, a histamine H2 receptor antagonist, to stimulate or inhibit secretion, respectively. F-actin staincd by rhodamine-phalloidin was seen along the apical and basolateral cell surface and in the intracellular canaliculi (IC) . The canalicular staining was more pronounced in gastrin-treated than in famotidine-treated rats. By immunofluerescence microscopy, fodrin was observed along the baso1ateral cell membrane and around IC in famotidine-treated rats; ankyrin coexisted with fodrin only in the basolateral cell membrane. Following treatment with gastrin IC was expanded and fodrin labeling in IC was less intense. Immunoelectron microscopy showed that fodrin and ankyrin were distributed along the baso1ateral cell membrane. Fodrin was also seen densely around IC, which distribution became sparser in stimulated cells. Neither fodrin nor ankyrin was found in the tubulovesicles. The present study showed that distribution of both F-actin and fodrin in IC changed according to the cell's secretory state and this chage might be related to the morphological alterations of the gastric parietal cell during secretion. In addition, coexistence of fodrin and ankyrin was revealed along the basolateral cell membrane.
We examined lectin binding sites in rod photoreceptor (RP) cells and inter- photoreceptor matrix (IPM) of Wistar rat retinas in situ using frozen thin-sections reacted with various labeled lectins. Neuraminidase digestion was employed to study the glycosyl residues next to the terminal sialic acid. Lectins used in this study are as follows: concanavalin A (Con A), wheat germ agglutinin (WGA), Maclura pomifera agglutinin (MPA), peanut agglutinin (PNA) and Dolichos biflorus agglutinin (DBA) . We also used isolated rod outer segments to study MPA-binding sites of the plasma membrane of the rod outer segment (ROS). In fluorescence micrographs of Con A- or WGA-fluorescence isothiocyanate (FITC) reacted retina, an intense staining was observed in the ROS and a meshwork-like staining was observed in the rod inner segment (RIS) and the outer nuclear layer. Electron microscopic observations of frozen thin-sections labeled with Con A- or WGA-ferritin revealed that these ligands were bound to disk membranes of the ROS, membranous structures of the RIS, entire plasma membranes of the RP cells, and the IPM. In MPA-FITC reacted retina, an intense staining in the ROS and the RIS showed a linear pattern. MPA-ferritin particles were bound to plasma membranes of the ROS and RIS, and to the IPM. In electron micrographs of isolated rod outer segments labeled with MPA-ferritin, MPA-bindings to the plasma membrane were greatly reduced, which suggest MPA was mainly bound to the IPM. No PNA- or DBA-labeling was observed in either RP cells or IPM. Following neuraminidase digestion, WGA-labeling was reduced and PNA-labeling appeared complementarily in the plasma membrane of the ROS and RIS, and the IPM.
The immunohistochemical localization of xanthine dehydrogenase in chicken liver was examined by indirect immunostaining, using anti-chicken xanthine dehydrogenase antisera and peroxidase-labeled anti-rabbit gamma-globulin. The enzyme activity of xanthine dehydrogenase in isolated hepatocytes was assayed by the biochemical method. Immunostaining of hepatic tissue sections showed that the cytoplasm of hepatocytes, Kupffer cells and endothelial cells lining the hepatic sinusoid was intensely stained. In chickens fed with a standard food containing 20% protein, the specific activity of 100, 000xg supernatant fraction of homogenate of isolated hepatocytes was almost the same as that of the whole liver. When the chickens were fed on a protein-rich diet, the enzyme activity in supernatant of homogenized isolated hepatocytes was 2.3 times higher than that in normal liver. Both the immunohistochemical and biochemical data in this study are comparable to each other, and suggest that xanthine dehydrogenase mainly exists in hepatocytes.
Localization of fodrin and α-actinin in the rat retinal pigment epithelium (RPE) in vivo was examined by immunolabeling of frozen sections as well as en face preparations. Fodrin was found densely in the apical cytoplasm and in the basal infolding; it was sparse in the lateral cell membrane and hardly present in the microvillus. On the other hand, α-actinin was concentrated in the dense material underlying the zonula adherens. Notably the protein was also seen along the microfilament bundle arising from the junction; it was present continuously in the vicinity of the junction, but localized only in electron-dense bodies deep in the cytoplasm. Fodrin was seen on both sides of the bundle but not in the bundle itself. The present results showed that the distribution of fodrin and α-actinin in RPE is distinctive among simple epithe1ia; fodrin is more concentrated in the basal cell membrane than in the lateral cell membrane; α-actinin is present in the actin filament bundle running through the apical cytoplasm and not in the terminal web. These differences are likely to be related to the specialized function and location of RPE.
Localization of α-actinin in the rat gastric parietal cell was studied using Lowicryl K4M embedding method as well as cryoultramicrotomy in combination with immunogold technique. These two methods gave similar results, but labeling efficiency was a little higher in cryoultrathin sections than in Lowicryl K4M sections. Labeling for α-actinin occurred in the cytoplasm adjacent to the zonula adherens, around the secretory canaliculus, in the microvillus, and along the basolateral infolding. Little labeling was seen around the tubulovesicle. Quantitative analysis of the distribution of gold particles in Lowicryl K4M sections revealed a significant decrease of α-actinin in the cytoplasm around the canaliculus in the secreting cell in comparison to the resting Cell. The present study showed that the gastric parietal cell is different from the intestinal epithelial cell in that it contains α-actinin in the microvilli. The decrease of α-acrinin around the intracellular canaliculus in the secretory state may be related to the morphological alteration of the parietal cell through reorganization of the actin filament meshwork.
Distribution of α-tubulin in rat-incisor odontoblasts and ameloblasts was observed by means of the immunohistochemical method. Wistar rats were fixed by perfusion with formalin solution or glutar-paraformaldehyde, and their incisors were dissected out, demineralized with either a solution of formic-acid formalin or EDTA, and prepared into 3μm-thick paraffin sections. The sections were incubated with a primary antibody (an anti-α-tubulin mouse monoclonal antibody) and then with a secondary antibody (a horseradish peroxidase-labeled sheep antimouse antibody). Intense and homogeneous α-tubulin immunostaining occurred in the cytoplasm of odontoblasts, inner enamel epithelial cells, and ameloblasts. In the odontoblast processes, however, the staining decreased gradually until it disappeared at a certain level in the dentin. The intense staining was noticed in the distal portions and Tomes' processes of differentiating and secreting ameloblasts. The staining generally decreased in the ameloblasts at an early stage of enamel maturation, and the cells frequently showed either slight or no staining. In reduced ameloblasts, the staining increased and returned to the level of the inner enamel epithelial cells. These results suggest that changes in their α-tubulin distribution reflect functional and morphological changes in odontoblasts and ameloblasts.
In the present investigation we have sought for atrial natriuretic peptide (ANP) -like immunoreactivity in aorta, carotid artery, cultured aortic cells and cultured cardiac myocytes of the rat at the light- and electron-microscopic level using antibodies directed against atriopeptin II (5-27) . Aortic and carotid aretery sections as well as cultured aortic cells did not show any ANP-specific immunoreactivity, preabsorbable by atriopeptin II or a-rat ANP (1-28) . Cultured cardiac myocytes could be shown to contain ANP granules and secrete immunoreactive ANP.
The coexistence of reduced nicotinamide adenine dinucleotide phosphate diaphorase (NADPH-d) with somatostatin (SOM) and choline acetyltransferase (ChAT) was compared between the rat neostriatum (ST) and basal forebrain complex (BF) . NADPH-d coexists with SOM in ST, but not in the ventral pallidum (VP), in the substantia innominata-nucleus basalis complex (SI-NB) and in the magnocellular preoptic nucleus (NPM) of the BF. In the medial septal nucleus as well as in the vertical and the horizontal limbs of the diagonal band (MS-vDB, hDB) representing the rostral BF and in the SI-NB, a high number of ChAT-positive cells also contain NADPH-d. The NPM shows a smaller number of double-stained cholinergic neurons. The functional significance of the NADPH-d coexistence in BF cholinergic cells is still unknown.
In the gallbladders of the guinea pig and mouse, the localizations and histochemical properties of certain connective tissue antigens were studied by means of indirect immunoperoxidase methods in light microscopy. These antigens were types I, III and IV collagens and fibronectin. The results obtained show that the four antigens exhibited such histochemical natures as are characteristic to each of them. The histophysiological significances of these antigens are discussed with special reference to the known physiological functions of the gallbladder to regulate biliary pressure throughout the bile drainage system of mammals.
In an attempt to characterize the membrane-associated Ca2+-ATPase activity in the roof and floor plates of the developing rat spinal cord (36), the effect of quercetin, a potent inhibitor of Ca2+-transporting ATPase was histochemically examined. Histochemical localization of ADPase and AMPase actvity was also investigated in relation to possible involvement in the enzymatic hydrolysis of ATP. When tissue specimens were incubated in media containing quercetin at a concentration of 0.1 or 0.5mM, the Ca2+-ATPase activity in both roof and floor plates appeared to be diminished, in a concentration-dependent manner, as seen under light microscopy. The inhibitory effect of quercetin was clearly evident in the floor plate. ADPase activity was also noted in both the roof and floor plates, particularly in the roof plate. Electron microscopic study revealed that the ADPase activity was mainly localized in the lateral and luminal plasma membranes of these plate-forming cells, resembli g the cytochemical localization of Ca2+-ATPase activity. There was, howover, no evidence of histochemical activity of AMPase in the developing spinal cord. These findings indicate that, in addition to activity of Ca2+-transporting ATPase, the roof and floor plate-forming cells during embryonic development may possess ecto-nucleotidases which hydrolyse ATP to AMP.