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 A₂ and phospholipase Cγ1

Abstract.

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 A₂ (PLA₂) and phospholipase Cγ1 (PLCγ1). The combination of immunogold labeling and KMnO₄–uranyl acetate–lead citrate staining for zymogenic contents clearly demonstrated the rapid diffusion of PLA₂ molecules from the exocytosed zymogenic contents into the mucinous contents in gastric glandular lumens. In contrast, the exocytosed PLCγ1 molecules remained within the zymogenic contents in the glandular lumens. These findings indicated the distinction between the exocytosed PLA₂ and PLCγ1 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 PLA₂ 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.