Gap Junctions and Ca2 Wave Propagation

The movement of cytoplasmic messengers between connected cells is mediated by gap junctions, which are clusters of intercellular channels that form a cytoplasmic bridge between adjacent cells to allow for the cell-to-cell transfer of ions, metabolites, and small messenger molecules. This includes Ca2+, ATP, cAMP, cADP-ribose, and IP3. Vertebrate intercellular channels are made up of a multigene family of conserved proteins called connexins. To date, at least 20 connexin genes have been identified. Connexins are made up of four hydrophobic transmembrane domains with the N- and C-termini located in the cytoplasm. In the plasma membrane, six connexin subunits assemble in a circle to form hemichannels called con-nexons. Connexons can contain a single type of con-nexin (homomeric) or multiple connexins (hetero-meric) to form the hemichannel pore. When two connexons from adjacent cells come together, they form an intercellular channel that spans the gap between the two cells. Two identical connexons or different connexons can join to form either homotypic or heterotypic intercellular channels, respectively. The presence of heteromeric connexins and hetero-typic intercellular channels can produce a diverse group of structurally distinct intercellular channels, with different permeabilities and/or functions. A variety of other factors, including membrane potential, Ca2+, pH, and phosphorylation of channels can also alter gap junction channels. Several neurotransmitters and hormones, such as dopamine, acetylcholine, GABA, and estrogens have also been found to alter intercellular channel activity.

Gap junctions are found in a variety of cell types and serve many functional roles, including normal and abnormal brain functions, wound healing, ciliary beating, bile flow, insulin secretion, follicular growth, oogenesis, ovulation, and bioluminescence. Mutations or defective productions in gap junction channels have been linked to a number of diseases, including Charcot-Marie-Tooth X-linked neuropathy, malignancy, infertility, cataractogenesis, and deafness.

At the cellular level, several lines of evidence implicate gap junction channels in mediating the propagation of intercellular Ca2+ waves. In airway epithelial cells, osteoblastic cells, endothelial cells, and astrocytes, the specific gap junction channel antagonists, octanol and halothane, inhibit the propagation of intercellular Ca2+ waves. These antagonists also prevented intercellular Ca2+ waves between Obelia photocytes and their support cells and between follicular cells and oocytes. In several cell lines, transfec-tion of cDNAs encoding gap junction proteins enabled them to propagate intercellular Ca2+ waves when they were not previously able to do so. For example, intercellular Ca2+ waves were observed in C6 glioma and HEK293 cells lines only after transfection with the cDNA for connexin 43. Agents that modify gap junction channels, such as H+, [Ca2+]i, and PKC, also alter the propagation of intercellular Ca2+ waves. Finally, the injection of specific antibodies against gap junction channels inhibited wave propagation in airway epithelial cells.

Calcium used for the generation of intercellular Ca2+ waves via gap junctions can come from two sources, intracellular Ca2+ stores and/or the intercellular space. In epithelial cells, aortic endothelial cells and mammary epithelial cells, removal of extracellular Ca2+ did not prevent the spread of a mechanically induced intercellular Ca2+ wave. Conversely, depletion of intracellular Ca2+ stores with thapsigargin prevents intercellular Ca2+ wave propagation. This indicates that intracellular Ca2+ stores are primarily responsible for the generation of Ca2+ waves in these cells. In other cells, removal of extracellular Ca2+ prevented Ca2+ wave propagation. Moreover, in some cells, the source of Ca2+ depends on the trigger for activation of intercellular Ca2+ waves. In glial cells, for example, glutamate-triggered Ca2+ waves are dependent on extracellular Ca2+ entry, whereas focal mechanical stimulation is dependent only on intracel-lular Ca2+ stores. In addition, unlike glutamate-stimu-lated waves, mechanically stimulated waves exhibit short delays at cell borders. These results demonstrate that different pathways for the generation of intercellular Ca2+ waves can exist in a single cell type.

Due to the nonselectivity of gap junctions to ions and small molecules, several diffusible second messenger molecules are potential candidates for mediating the propagation of intercellular Ca2+ waves via gap junctions. Because many Ca2+-mobilizing receptors stimulate the generation of intercellular Ca2+ waves, two obvious candidates are Ca2+ and IP3. One model for the propagation of Ca2+ waves suggests that there is a regenerative interaction between [Ca2+]i and the release of additional Ca2+ from intracellular Ca2+ stores. Thus, Ca2+ diffuses short distances to stimulate the autocatalytic release of Ca2+ from intracellular stores to propagate the intercellular Ca2+ wave. Consistent with the involvement of Ca2+ diffusion in the generation of the intercellular Ca2+ wave, addition of exogenous Ca2+ buffers to the cytoplasm of glial cells slows or abolishes intercellular Ca2+ waves. One uncertainty with this model, however, is that it remains unknown if Ca2+ diffuses through gap junctions to stimulate the release of Ca2+ in the adjoining cell. A further complication is that in some cells bathed in Ca2+-deficient medium, there is no increase in [Ca2+]i in the stimulated cell, yet a Ca2+ wave occurs in adjacent cells. This indicates that an increase in [Ca2+]i in the stimulated cell is not required for the initiation of intercellular Ca2+ waves. Thus, Ca2+ is not the cytosolic messenger that propagates Ca2+ waves through gap junctions in these cells.

Another model for the propagation of intercellular Ca2+ waves involves the regenerative production of IP3. In this model, it is suggested that the rise in [Ca2+]i stimulated by IP3 activates phospholipase C to generate additional IP3. Consistent with the involvement of IP3 in the generation of intercellular Ca2+ waves, inhibiting IP3 synthesis or blocking its receptors eliminates the propagation of Ca2+ waves. In addition, inhibition of phospholipase C abolishes mechanically stimulated waves in some cells, pointing to its involvement in the regenerative generation of IP3. Mathematical modeling simulations indicate that intercellular wave propagation cannot be explained by the simple diffusion of IP3, as the required concentration for diffusion is much higher than that reached by agonist stimulation. This further supports the hypothesis that regenerative IP3 production mediates the propagation of intercellular Ca2+ waves. As IP3 molecules are small enough to pass through gap junctions, the simplest hypothesis to explain IP3-mediated intercellular Ca2+ waves is that IP3 is the intercellular diffusible messenger that synchronizes cellular activity by the production of intercellular Ca2+ waves.

In addition to IP3 and Ca2+, cADP-R is also a good candidate for mediating intercellular Ca2+ waves. Like IP3 and Ca2+, molecules of cADP-R are small enough to diffuse through gap junctions, indicating that they could trigger the intercellular Ca2+ wave in adjoining cells. In addition, like IP3, cADP-R stimulates Ca2+ release in a variety of cell types and can act as a global messenger within single cells. Accordingly, in sheep lens cells, injection of cADP-R stimulates the production of intercellular Ca2+ waves.

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