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 Cgamma 1

High-pressure freezing/freeze substitution followed by Lowicryl K4M embeddi ng provided an excellent morphology and antigenicity of the gastric glands, as well as the intraluminal fluid contents. Taking advantage of this, we h istochemically investigated the secretory dynamics of the zymogenic content s in rat gastric gland, with special references to phospholipase A(2) (PLA( 2)) and phospholipase C gammal (PLC gammal). The combination of immunogold labeling and KMnO4-uranyl acetate-lead citrate staining for zymogenic conte nts clearly demonstrated the rapid diffusion of PLA(2) molecules from the e xocytosed zymogenic contents into the mucinous contents in gastric glandula r lumens. In contrast, the exocytosed PLC gammal molecules remained within the zymogenic contents in the glandular lumens. These findings indicated th e distinction between the exocytosed PLA(2) and PLC gammal in their diffusi on rate. In addition, the mucinous contents surrounding the exocytosed zymo genic contents were intensely labeled with Griffonia simplicifolia II lecti n which specifically recognizes the mucin of mucous neck cells. Interesting ly, some of the PLA(2) immunolabeling on the mucinous contents was associat ed with the apical membranes of gastric epithelial cells, especially that o f parietal cells. The secretory dynamics of the zymogenic contents in rat g astric glands, including their interaction with the mucinous contents are d iscussed.