No abstract

Abstract. Analysis by SDS-PAGE of gap junction fractions isolated from heart suggests that the junctions are comprised of a protein with an Mr 43,000. Antibodies against the electroeluted protein and a peptide representing the 20 amino terminal residues bind specifically on immunoblots to the 43-kD protein and to the major products arising from proteolysis during isolation. By immunocytochemistry, the protein is found in ventricle and atrium in patterns consistent with the known distribution of gap junctions. Both antibodies bind exclusively to gap junctions in fractions from heart examined by EM after gold labeling. Since only domains of the protein exposed at the cytoplasmic surface should be accessible to antibody, we conclude that the 43-kD protein is assembled in gap junctions with the amino terminus of the molecule exposed on the cytoplasmic side of the bilayer, that is, on the same side as the carboxy terminus as determined previously. By combining proteolysis experiments with data from immunoblotting, we can identify a third cytoplasmic region, a loop of some 4 kD between membrane protected domains. This loop carries an antibody binding site. The protein, if transmembrane, is therefore likely to cross the membrane four times. We have used the same antisera to ascertain if the 43-kD protein is involved in cell-cell communication. The antiserum against the amino terminus blocked dye coupling in 90% of cell pairs tested; the antiserum recognizing epitopes in the cytoplasmic loop and cytoplasmic tail blocked coupling in 75 % of cell pairs tested. Preimmune serum and control antibodies (one against MIP and another binding to a cardiac G protein) had no or little effect on dye transfer. Our experimental evidence thus indicates that, in spite of the differences in amino acid sequence, the gap junction proteins in heart and liver share a general organizational plan and that there may be several domains (including the amino terminus) of the molecule that are involved in the control of junctional permeability.

We have compared intercellular communication in the regenerating and normal livers of weanling rats . The electrophysiological studies were conducted at the edge of the liver, and we have found that here as elsewhere in the liver there is a dramatic decrease in the number and size of gap junctions during regeneration . The area of hepatocyte membrane occupied by gap junctions is reduced 100-fold 29-35 h after hepatectomy . By combining observations made with the scanning electron microscope with our freeze fracture data we have estimated the number of "communicating interfaces" (areas of contact between hepatocytes that include at least one gap junction) formed by hepatocytes in normal and regenerating liver. In normal liver a hepatocyte forms gap junctions with every hepatocyte it contacts (-6) . In regenerating liver a hepatocyte forms detectable gap junctions with, on average, only one other hepatocyte .Intercellular spread of fluorescent dye and electric current is reduced in regenerating as compared with normal liver. The incidence of electrical coupling is reduced from 100% of hepatocyte pairs tested in control liver to 92% in regenerating liver. Analysis of the spatial dependence of electrotonic potentials indicates a substantial increase in intercellular resistance in regenerating liver. A quantitative comparison of our morphological and physiological data is complicated by tortuous pattern of current flow and by inhomogeneities in the liver during regeneration, Nevertheless we believe that our results are consistent with the hypothesis that gap junctions are aggregates of channels between cell interiors. Cells in most vertebrate tissues, in embryos, in tissue culture, and in many invertebrates are interconnected by low-resistance pathways permeable to ions and small molecules. Generally speaking, this pathway behaves as a simple aqueous tunnel between cell interiors (see references 5 and 33 for recent reviews). There is strong, but as yet only circumstantial evidence that the gap junction is the site of this intercellular pathway .Early evidence for the role of specialized cell junctions in electrical coupling between excitable cells came from the work of Barr et al (2,3,12) and Dreifuss et al (13). Potter et al (46) as well as Loewenstein and Kanno (34) obtained evidence for such junctions in nonexcitable tissues. Studies of the electrotonic synapses of the crayfish medial giant axon showed that increases in the junctional resistance between axon segments THE JOURNAL OF CELL BIOLOGY " VOLUME 91 NOVEMBER 1981 505-523 © The Rockefeller University Press " 0021-9525/81/11/0505/19 $1 .00were associated with the disappearance of gap junctions between axonal membranes (41). Subsequently, two studies specifically associated the presence of gap junctions with intercellular communication . In the brown fat of newborn mice and in baby hamster kidney (BHK) cells grown in tissue culture, systems in which electrotonic coupling had already been demonstrated (20, 52), Revel et al (49) showed that gap junctio...

No abstract

Abstract. There is a reduction in the 28-kD gap junction protein detectable by immunofluorescence in livers of partially hepatectomized rats and in cultured hepatocytes stimulated to proliferate. By the coordinate use of antibodies directed to the hepatic junction protein (HJP28) and the use of a monoclonal antibody that recognizes bromodeoxyuridine (BrdU) incorporated into DNA, we have been able to study the relationship between detectable gap junction protein and cell division. Hepatocytes that label with BrdU in the regenerating liver and in cell culture show a significant reduction of HJP28. Cells that do not synthesize DNA, on the other hand, show normal levels and distribution of immunoreactive gap junction protein. We postulate that the quantitative changes in gap junction expression might play an important role in the control of proliferation in the liver.AP junctions represent a class of intercellular contacts which are commonly assumed to be the site of exchange of inorganic ions and small molecules (3,4,14,35). It has been suggested that they may be involved in regulating various cellular activities such as growth control (7,27), and the modulation of developmental processes during embryogenesis (19,38,39,44). There is a close correlation between the ultrastructural demonstration of gap junctions and the presence of cell-cell coupling measured electrophysiologically or by injection of fluorescent dyes (2, 14, 36). Several laboratories have recently reported the characterization of antibodies directed against the 28-kD protein that is a major component of isolated rat liver gap junctions (hepatic junction protein, HJP28) ~ (9,20,23,24,32,42). Intracellular injection of specific antibodies raised against rodent HJP28 inhibits electrical or dye-coupling between several types of vertebrate cells (21, 28). Injection of antibodies raised against this protein into blastomeres of Xenopus appears to cause developmental defects in the larva (43). These antibodies thus detect a functionally competent form of HJP28.The regenerating liver is an interesting experimental model where the possible correlation between gap junctions and the control of growth can be examined (12,29,41,45,46). Partial hepatectomy causes a burst of cell division that, by morphometric studies of freeze-cleaved material, has been shown to be temporally correlated with a cycle of disappearance and reappearance of gap junctions (45, 46). A significant reduction of the HJP28 has also been seen after partial hepatectomy by biochemical and immunochemical criteria (12, 41). Abbreviations used in this paper:BrdU, bromodeoxyuridine; HJP, hepatic junction protein.Not all cells participate in this wave of cell proliferation (34) nor is there a complete loss of junctional communication (29). Until now it has not been possible to establish a definite link between cell division and the loss of gap junction. The experiments described here use a double-immunolabeling approach to the study of regenerating liver cells and of cultured primary hepatocytes from...