We have studied the cffccts of phospholipasc C from Clostridium welchii on gap junctions in the intact mouse liver and in a junction-rich fraction prepared from mouse liver. Treatment of the isolated junctions results in the disappearance of both the 20 A gap and of thc polygonal lattice visible with lanthanum. The junctions are morphologically unaltered, however, when whole livers are perfused with phospholipasc via the portal vein. These results suggest that cxtracellular phospholipase cannot diffuse into the junctional area, but that the enzyme may affect structures within the gap from its cytoplasmic surfaces which become exposed in the isolated preparations. Horseradish peroxidase, which has physical dimensions similar to those of Clostridium phospholipase is also denied access to the 20 A gap in whole liver, while pcroxidase reaction product can be seen in the gap in isolated preparations. Beef liver catalase, however, a tracer molecule much larger than peroxidase, cannot penetrate even in isolated fractions. If the cytoplasmic approaches to the gap junction used by peroxidase and phospholipase arc available in vivo, and have not been created during the process of mechanical isolation, thcy may play a role in cell-to-cell passage of molecules larger than ions. I N T R O D U C T I O N The gap junction in mouse liver, which appears in the electron microscope as an area of intimate cell-to-cell apposition, has been previously described in detail (8, 24). Use of colloidal lanthanum reveals a polygonal lattice of substructures in the 20 A gap which separates the outer leaflets of the apposed junctional membranes (24). The polygonal lattice may also be visualized after negative staining of isolated membrane preparations (1, 2), and by the freeze-cleave technique (11). In an earlier paper (8), we provided evidence that the appearance of the 20 A gap and associated polygonal lattice could be altered by exposure to polar organic solvents, notably acetone. At a critical concentration of 60% aqueous acetone, both the 20 A gap and the polygonal lattice disappeared from thin-sectioned, freeze-cleaved, and negatively stained material. Thin-layer chromatography revealed that a complex group of phospholipids were solubilized by the acetone at the critical concentration. To further investigate the role of phospholipids, we have studied the effects of phospholipase C from Clostridium welchii on the gap junction. The phospholipase, however, is a much larger molecule than the organic solvents which were used previously. As a control we have therefore made a parallel study with horseradish peroxidase, a THE JOURNAL OF CELL BIOLOGY • VOLUME 50, 1971 • pages 81-91 81 molecule of physical dimensions similar to Clostridium phospholipase C, and with catalase, a much larger molecule. The peroxidase serves as a tracer, out l ining the routes avai lable for the penet ra t ion of phospholipase into the gap junct ion. M A T E R I A L S A N D M E T H O D S Mature mice obtained from the Charles River Breeding Labs., Inc., Wilmington, Mass., were used throughout. Fixation and Embedding All aldehyde fixation was carried out in 3% glutaraldehyde in 0.2 M s-collidlne (3). Alcian blue 8GX (Allied Chemical Corp., New York.) was added to this fixative in 0. 1% concentration when used (26). Postfixation was done in a 2:1 mixture of 2% aqueous OsO4 and collidine. Whole livers were fixed by perfusion as previously described (8). The livers were kept warm with an infrared lamp during the phospholipase perfusions. For the horseradish peroxidase experiments, livers were fixed by immersion at room temperature, The livers were cut into 1 mm cubes and stained en bloc with 2% uranyl acetate for l hr (10) and then rapidly dehydrated in ethanol and embedded in Epon-Araldite (16). Silver sections were examined in a Siemens Elmiskop 1A.
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