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Hemichannels in the overlapping regions of apposing cells plasma membranes join to form gap junctions and provide an intercellular communication pathway. Hemichannels are also present in the nonjunctional regions of individual cells and their activity is gated by several agents, including calcium. However, their physiological roles are unknown. Using techniques of atomic force microscopy (AFM), fluorescent dye uptake assay, and laser confocal immunofluorescence imaging, we have examined the extracellular calcium-dependent modulation of cell volume. In response to a change in the extracellular physiological calcium concentration (1.8 to

More than one isotype of the gap junction channel-forming protein subunit, known as connexin, are synthesized in most mammalian cells. Using a modified primary cell culture of rat hepatocytes, in which both connexin32 and connexin26 were expressed in a comparable degree, the molecular composition of connexin subtypes in a gap junction channel (i.e., homomeric or heteromeric) was studied. A fluorescent dye Lucifer Yellow-coupling among hepatocytes was blocked in the presence of 20 microM beta-glycyrrhetinic acid, and antagonist for the gap junction channel. Similarly, either one of the antibodies raised against connexin32 or connexin26 completely inhibited dye-coupling activity among hepatocytes. In addition, we studied the dye-coupling properties at different extracellular pHs in two primary rat hepatocytes: one in which connexin26 was induced, and the other which expresses connexin32 as a major gap junction channel protein. The dye-coupling among the connexin26-induced hepatocytes was more sensitive to lowering pHs than among the normal hepatocytes, in which less than 5% of gap junction protein is connexin26. Taken together, data obtained in our study strongly suggest that the connexins (32 & 26) are assembled to form heteromeric, rather than homomeric, gap junction channels in the connexin226-induced rat hepatocytes.

Intercellular connexin channels (gap junction channels) have long been thought to mediate molecular signaling between cells, but the nature of the signaling has been unclear. This study shows that connexin channels from native tissue have selective permeabilities, partially based on pore diameter, that discriminate among cytoplasmic second messenger molecules. Permeability was assessed by measurement of selective loss/retention of tracers from liposomes containing reconstituted connexin channels. The tracers employed were tritiated cyclic nucleotides and a series of oligomaltosaccharides derivatized with a small uncharged fluorescent moiety. The data define different size cut-off limits for permeability through homomeric connexin-32 channels and through heteromeric connexin-32/connexin-26 channels. Connexin-26 contributes to a narrowed pore. Both cAMP and cGMP were permeable through the homomeric connexin-32 channels. cAMP was permeable through only a fraction of the heteromeric channels. Surprisingly, cGMP was permeable through a substantially greater fraction of the heteromeric channels than was cAMP. The data suggest that isoform stoichiometry and/or arrangement within a connexin channel determines whether cyclic nucleotides can permeate, and which ones. This is the first evidence for connexin-specific selectivity among biological signaling molecules.

This study was conducted to assess the effects of chemical carcinogens on the gap junction-mediated intercellular communication in cultured mammalian cells. The method of scrape-loading dye transfer of lucifer yellow was adapted as a measure of gap junctional communication. Clone 9 cells derived from rat liver were treated with a model chemical carcinogen, N-methyl-N'-nitro-N-nitrosoguanidine (MNNG) and the gap junctional communication was assessed by measuring the transfer of scrape-loaded lucifer yellow dye. When cells were treated with the carcinogen at 0.3 mg/ml, the fluorescent dye transfer was inhibited by 90% in 60 min. Other chemical agents, which include direct or indirect carcinogens and antitumor drugs, were also examined for their effects on the gap junctional communication. Direct carcinogens, such as MNNG, hydroxylamine and ethidium bromide, exhibited strong inhibition of intercellular communication, while indirect carcinogens, such as aflatoxin B1 and ethionine, exerted minor effects. Effects of test chemicals on the cell communication through gap junctions were readily quantitated by counting the number of cells stained with the fluorescent dye.