Advanced glycation end-products (AGEs) result from oxidation-reduction reactions that ensue when a sugar becomes adducted to a protein. AGEs cause various complications of diabetes mellitus (DM). Experimental and clinical evidence suggest that AGEs also contribute to the complications of hypertension (HTN). Little is known about the abundance and localization of AGEs in human myocardium. In a few light microscopic studies, the AGE carboxymethyl lysine (CML) has been immunolabeled and localized virtually exclusively to the walls of small arteries. To more precisely delineate the abundance and localization of CML, we developed an immunoelectron microscopic (IEM) detection method using anti-CML monoclonal antibody 6D12 in conjunction with computer-assisted image analysis. Antibody was pre-absorbed with purified AGE-bovine serum albumin to assure specificity. Antigen-antibody (ag-ab) complexes were individually identified with protein A-conjugated colloidal gold and counted with an automated system. We applied this method in 21 patients (pts) undergoing epicardial biopsy during coronary bypass grafting (CBG) [20 M, 1 F; mean age 65 +/- 7.4 (+/- SEM) years]. Seven pts had neither DM nor HTN, seven had HTN, and seven had DM + HTN. In contrast to the prior light microscopic studies, we detected CML scattered throughout the cardiomyocyte in all pts, but in widely varying amounts. Ag-ab complexes were abundant in sections through myofilaments (mean count 23.6 +/- 9.2 per microm(2), range 9.4-48) and even more so in mitochondria (mean count 34.4 +/- 11.9 per microm(2), range 14.1-68.2, P < 0.001 vs. myofilaments). CML was also detected in vascular endothelial cells. There were no statistically significant differences based on presence or absence of HTN or DM. In conclusion, our IEM method is the first to provide detailed delineation of the localization and abundance of CML in myocardium. CML is very prevalent in CBG pts, suggesting that AGEs could play a role in abnormal cardiomyocyte function, including altered energy metabolism.

The regulation of platelet volume significantly affects its function. Because water is the major molecule in cells and its active transport via water channels called aquaporins (AQPs) have been implicated in cellular and organelle volume regulation, the presence of water channels in platelets and their potential role in platelet volume regulation was investigated. G-protein–mediated AQP regulation in secretory vesicle swelling has previously been reported in neurons and in pancreatic acinar cells. Mercuric chloride has been demonstrated to inhibit most AQPs except AQP6, which is stimulated by the compound. Exposure of platelets to HgCl2-induced swelling in a dose-dependent manner, suggesting the presence of AQP6 in platelets. Immunoblot analysis of platelet protein confirmed the presence of AQP6, and also of Gαo, Gαi–1 and Gαi–3 proteins. Results from this study demonstrate for the first time that in platelets AQP6 is involved in cell volume regulation via a G-protein–mediated pathway.

We studied the antibodies hepatocyte paraffin 1 (Hep Par 1) and thyroid transcription factor-1 (TTF-1; clone 8G7G3/1) in normal human liver tissue with immunoelectron microscopy using renal tubules as control samples. TTF-1 (8G7G3/1) and Hep Par 1 bound exclusively to the mitochondria of hepatocytes but not to those of the renal tubular epithelium. Both antibodies labeled mitochondria in a similar pattern and density. These findings confirm that the binding site of Hep Par 1 in hepatocytes is mitochondrial. The specific binding of TTF-1 (8G7G3/1) in hepatocyte mitochondria suggests its potential usefulness for identifying hepatocellular carcinoma. Western blot analysis with cellular proteins extracted from normal human liver and thyroid tissue demonstrated that Hep Par 1 and TTF-1 (8G7G3/1) bound to a protein band of approximately 150 kd in liver cells, with TTF-1 (8G7G3/1) showing less affinity. It is likely that different epitopes to Hep Par 1 and TTF-1 (8G7G3/1) share the same protein molecule in hepatocyte mitochondria.