Abstract. The tight junction is an essential element of the intercellular junctional complex; yet its protein composition is not fully understood. At present, only three proteins, ZO-1 (Stevenson, B. R., J. D. Siliciano, M. S. Mooseker, and D. A. Goodenough. 1986. J. Cell Biol. 103:755-766), cingulin (Citi, S., H. Sabanay, R. Jakes, B. Geiger, and J. Kendrick-Jones. 1988. Nature (Lond.). 333:272-275) and ZO-2 (Gumbiner, B., T. Lowenkopf, and D. Apatira. 1991. Proc. Natl. Acad. Sci. USA. 88:3460-3464) are known to be associated with the tight junction. We have generated a monoclonal antibody (7H6) against a bile canaliculusrich membrane fraction prepared from rat liver. This 7H6 antigen was preferentially localized by immunofluorescence at the junctional complex regions of hepatocytes and other epithelia, and 7H6-aftiliated gold particles were shown electron microscopically to localize at the periphery of tight junctions. Immunoblot analysis of a bile canaliculus-rich fraction of rat liver using 7H6, anti-ZO-1 antibody (R26.4C), and anticingulin antibody revealed that 7H6 reacted selectively with a 155-kD protein, whereas R26.4C reacted only with a 225-kD protein. Anti-cingulin antibody reacted solely with 140 and 108-kD proteins, indicating that the protein recognized by 7H6 is immunologically different from ZO-1 and cingulin. Immunoprecipitation of detergent extracts obtained from metabolically labeled MDCK cells with R26.4C coprecipitated a 160-kD protein, which corresponds to ZO-2, with ZO-1. However, 7H6 did not react with the 160-kD protein.These results strongly suggest that the 7H6 antibody recognizes a novel tight junction-associated protein different from ZO-1, cingulin and ZO-2.

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The cardiomyopathic Syrian hamster develops focal myocardial necrosis beginning at 1 month of age, which leads to eventual ventricular failure within 1 year. The pathogenesis of this myocytolytic necrosis is unknown. Based on the nature of the cell necrosis, cytochemical evidence of vascular alterations, and the sensitivity of the hamsters to catecholamines and other vasoactive substances, we believe that the cardiomyopathy may be mediated by abnormalities of the microcirculation. Nonetheless, until the present study, no significant changes have been observed in these vessels. To elucidate the pathogenesis of this disease, we perfused living cardiomyopathic hamsters with silicone rubber solutions, which revealed numerous areas of microvascular constriction, diffuse vessel narrowing and luminal irregularity. Fixed structural lesions in these vessels could not be demonstrated. Pretreatment of young hamsters with verapamil during the period when they normally develop myocardial necrosis prevented myocytolytic lesions and abolished microvascular hyperreactivity. We believe that focal, transient spasm of small blood vessels, probably secondary to vasoactive substances, may cause myocytolytic necrosis (a form of reperfusion injury) in this model. This may also be a multifactorial disease with myocellular as well as vascular abnormalities leading to myocardial degeneration. The similarity of this disease to human and experimental cardiomyopathy suggests that microvascular spasm may be a common denominator of many different cardiomyopathic syndromes.

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