Analysis of gap junction formation in rat hepatocytes by intravenous injection of an anti-connexin32 monoclonal antibody (HAM8).

HAM8 monoclonal antibody was used to examine the mechanism of connexin32 (Cx32) formation in the rat model in vivo by immunofluorescence and immunoelectron microscopy. After a single intravenous injection of HAM8 IgG, a number of HAM8 signals were observed at the sinusoidal face, between adjacent hepatocytes as well as in the cytoplasm stained with only 2nd fluorescent antibody. Moreover, the in vivo localization of the HAM8 antigen appeared to change with time. At 5 min after the antibody injection, Cx32 signals from liver sections were clearly detected at the sinusoidal face. Fifteen minutes later, numerous linear and dotted fluorescent signals were observed between hepatocytes; in addition, much punctate staining was found at the sinusoidal face and in hepatocytes. These findings were identified by immunoelectron microscopy. Interestingly, 1 hr later, much punctate staining considered to be similar to those seen in the normal rat liver tissues was observed between adjacent hepatocytes, suggesting that a great deal of Cx32 combined with HAM8 have been assembled into identifiable gap junction plaques. Five hours later, intercellular and intracellular Cx32 signals were infrequently detected. When staining was performed with HAM8 and 2nd antibody, however, numerous Cx32 signals were again observed between neighboring hepatocytes, as punctate staining appearing in a pattern approximately the same as that seen in the normal liver tissue. Based on these results, we assumed that a precursor gap was present during Cx32 formation, and discussed the pathways of Cx32 formation and the degradation of Cx32 as well as that of HAM8.