No abstract

Recently, ATP has gained much interest as an extracellular messenger involved in the communication of calcium signals between cells. The mechanism of ATP release is, however, still a matter of debate. In the present study we investigated the possible contribution of connexin hemichannels or ion channels in the release of ATP in GP8, a rat brain endothelial cell line. Release of ATP was triggered by photoactivation of InsP(3) or by reducing the extracellular calcium concentration. Both trigger protocols induced ATP release significantly above baseline. InsP(3)-triggered ATP release was completely blocked by alpha-glycyrrhetinic acid (alpha-GA), the connexin mimetic peptides gap 26 and 27, and the trivalent ions gadolinium and lanthanum. ATP release triggered by zero calcium was, in addition to these substances, also blocked by flufenamic acid (FFA), niflumic acid, and NPPB. Gap 27 selectively blocked zero calcium-triggered ATP release in connexin-43 transfected HeLa cells, while having no effect in wild-type and connexin-32 transfected cells. Of all the agents used, only alpha-GA, FFA and NPPB significantly reduced gap junctional coupling. In conclusion, InsP(3) and zero calcium-triggered ATP release show major similarities but also some differences in their sensitivity to the agents applied. It is suggested that both stimuli trigger ATP release through the same mechanism, which is connexin-dependent, permeable in both directions, potently blocked by connexin mimetic peptides, and consistent with the opening of connexin hemichannels.

Connexin hemichannels, that is, half gap junction channels (not connecting cells), have been implicated in the release of various messengers such as ATP and glutamate. We used connexin mimetic peptides, which are, small peptides mimicking a sequence on the connexin subunit, to investigate hemichannel functioning in endothelial cell lines. Short exposure (30 min) to synthetic peptides mimicking a sequence on the first or second extracellular loop of the connexin subunit strongly supressed ATP release and dye uptake triggered by either intracellular InsP(3) elevation or exposure to zero extracellular calcium, while gap junctional coupling was not affected under these conditions. The effect was dependent on the expression of connexin-43 in the cells. Connexin mimetic peptides thus appear to be interesting tools to distinguish connexin hemichannel from gap junction channel functioning. In addition, they are well suited to further explore the role of connexins in cellular release or uptake processes, to investigate hemichannel gating and to reveal new unknown functions of the large conductance hemichannel pathway between the cell and its environment. Work performed up to now with these peptides should be re-interpreted in terms of these new findings.

No abstract

Astrocytes and endothelial cells are in close contact with each other at the blood-brain barrier, where important molecular transports take place. Despite these key morphological and functional properties, little is known regarding the dynamic signalling processes that occur between these two cell types. We investigated astrocyte-endothelial cell calcium signalling mechanisms in a coculture model prepared from primary rat cortical astrocytes and ECV304 cells. We used flash photolysis of caged inositol-trisphosphate (IP3) and gentle mechanical stimulation to trigger astrocyte-endothelial cell calcium signals and to investigate the underlying propagation mechanisms. Photolytically releasing IP3 in a single cell triggered increases in cytoplasmic calcium concentration that propagated between astrocytes and endothelial cells in either direction. These propagating calcium signals did not cross cell-free zones and were not affected by fast superfusion or by the purinergic inhibitors apyrase and suramin, indicating that they are communicated through an intracellular pathway in conjunction with gap junctions. Electrophysiological experiments confirmed a low degree of astrocyte-endothelial cell electrical cell-to-cell coupling. Mechanical stimulation of a single cell also triggered astrocyte-endothelial cell calcium signals but, in contrast to the former triggering mode, these signals crossed cell-free zones and were significantly inhibited by apyrase, thus indicating the involvement of an extracellular and purinergic messenger. Astrocyte-endothelial cell calcium signalling also occurred in cocultures prepared with astrocytes and primary rat brain capillary endothelial cells. We conclude that astrocytes and endothelial cells can exchange fast-acting calcium signals (time scale of seconds) that can be communicated through an intracellular/gap junctional pathway and an extracellular purinergic pathway.