Immunocytochemical demonstration of the secretory dynamics of zymogenic contents in rat gastric gland processed by high-pressure freezing/freeze substitution, with special references to phospholipase A(2) and phospholipase Cgamma1.

High-pressure freezing/freeze substitution followed by Lowicryl K4M embedding provided an excellent morphology and antigenicity of the gastric glands, as well as the intraluminal fluid contents. Taking advantage of this, we histochemically investigated the secretory dynamics of the zymogenic contents in rat gastric gland, with special references to phospholipase A(2) (PLA(2)) and phospholipase Cgamma1 (PLCgamma1). The combination of immunogold labeling and KMnO4-uranyl acetate-lead citrate staining for zymogenic contents clearly demonstrated the rapid diffusion of PLA(2) molecules from the exocytosed zymogenic contents into the mucinous contents in gastric glandular lumens. In contrast, the exocytosed PLCgamma1 molecules remained within the zymogenic contents in the glandular lumens. These findings indicated the distinction between the exocytosed PLA(2) and PLCgamma1 in their diffusion rate. In addition, the mucinous contents surrounding the exocytosed zymogenic contents were intensely labeled with Griffonia simplicifolia II lectin which specifically recognizes the mucin of mucous neck cells. Interestingly, some of the PLA(2) immunolabeling on the mucinous contents was associated with the apical membranes of gastric epithelial cells, especially that of parietal cells. The secretory dynamics of the zymogenic contents in rat gastric glands, including their interaction with the mucinous contents are discussed.

Multistratified expression of polysialic acid and its relationship to VAChT-containing neurons in the inner plexiform layer of adult rat retina.

We investigated the localization of polysialic acid (PSA), neural cell adhesion molecule (NCAM), and vesicular acetylcholine transporter (VAChT) in adult rat retina by using immunofluorescence with a confocal laser scanning microscope. Western blot analysis showed a typical broad smear of PSA and isoforms of NCAM (120, 140, and 180 kD). PSA immunofluorescence revealed multistratification in the inner plexiform layer (IPL). Dual immunostaining for PSA and NCAM exhibited the selective co-expression of PSA and NCAM on Müller cells. Moreover, dual immunolabeling for PSA and VAChT completely separated the five strata in the IPL. Strata 1, 3, and 5 were immunoreactive for PSA and Strata 2 and 4 for VAChT. These results suggest the possibility that PSA molecules on Müller cells are spatially related to ON and OFF retinal channels in the IPL.

Characterization of gastric Na+/I- symporter of the rat.

Characterization of gastric Na+/I- symporter (NIS) of the rat was carried out. Sequencing of the open reading frame of gastric NIS mRNA showed only three nucleotide changes when compared with FRTL-5 NIS cDNA, and two of these changes led to amino acid changes. The results of Northern blot analysis showed that abundant NIS mRNA was expressed in the stomach when compared with other organs. Western blot analysis using gastric mucosa and FRTL-5 lysates detected the difference in molecular weight between FRTL-5 and gastric mucosa lysates, suggesting abnormal posttranslational modification of gastric NIS protein. Immunohistochemically, gastric NIS protein was located in the cornification layer of the stratified squamous epithelium of the pars proventricularis and in parietal cells and on the apical border of surface epithelial cells of the pars glandularis. Gastric NIS protein was present in tubulovesicular structures and lysosomes in parietal cells by immunoelectron microscopy. Gastric NIS protein exists to trap I- from the gastric lumen, except in parietal cells. Results indicated that a very large amount of gastric NIS mRNA is expressed to be translated, whereas only a small amount of immature gastric NIS protein is detected. This may indicate that immature gastric NIS protein rapidly degrades to peptides.