Connexin hemichannels have been proposed as a diffusion pathway for the release of extracellular messengers like ATP and others, based on connexin expression models and inhibition by gap junction blockers. Hemichannels are opened by various experimental stimuli, but the physiological intracellular triggers are currently not known. We investigated the hypothesis that an increase of cytoplasmic calcium concentration ([Ca2+]i) triggers hemichannel opening, making use of peptides that are identical to a short amino-acid sequence on the connexin subunit to specifically block hemichannels, but not gap junction channels. Our work performed on connexin 32 (Cx32)-expressing cells showed that an increase in [Ca2+]i triggers ATP release and dye uptake that is dependent on Cx32 expression, blocked by Cx32 (but not Cx43) mimetic peptides and a calmodulin antagonist, and critically dependent on [Ca2+]i elevation within a window situated around 500 nM. Our results indicate that [Ca2+]i elevation triggers hemichannel opening, and suggest that these channels are under physiological control.

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.

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The communication of calcium signals between cells is known to be operative between neurons where these signals integrate intimately with electrical and chemical signal communication at synapses. Recently, it has become clear that glial cells also exchange calcium signals between each other in cultures and in brain slices. This communication pathway has received utmost attention since it is known that astrocytic calcium signals can be induced by neuronal stimulation and can be communicated back to the neurons to modulate synaptic transmission. In addition to this, cells that are generally not considered as brain cells become progressively incorporated in the picture, as astrocytic calcium signals are reported to be communicated to endothelial cells of the vessel wall and can affect smooth muscle cell tone to influence the vessel diameter and thus blood flow. We review the available evidence for calcium signal communication in the central nervous system, taking into account a basic functional unit -the brain cell tripartite-consisting of neurons, glial cells and vascular cells and with emphasis on glial-vascular calcium signaling aspects.

The breaching of the blood-brain barrier is an essential aspect in the pathogenesis of neuroinflammatory diseases, in which tumour necrosis factor alpha (TNF-a) as well as endothelial calcium ions play a key role. We investigated whether TNF-a could influence the communication of calcium signals between brain endothelial cells (GP8 and RBE4). Intercellular calcium waves triggered by mechanical stimulation or photoliberation of InsP 3 in single cells were significantly reduced in size after TNF-a exposure (1000 U/mL, 2 and 24 h). Calcium signals are communicated between cells by means of gap junctional and paracrine purinergic signalling. TNF-a significantly inhibited gap junctional coupling, stimulated the basal release of ATP, and dose-dependently blocked the triggered component of ATP release. The cytokine displayed similar effects on the uptake of a fluorescent reporter dye into the cells. Previous work with connexin mimetic peptides demonstrated that the triggered ATP release in these cells is connexin-related; these peptides did, however, not influence the elevated basal ATP release caused by TNF-a. We conclude that TNF-a depresses calcium signal communication in blood-brain barrier endothelial cells, by reducing gap junctional coupling and by inhibiting triggered ATP release. The cytokine thus inhibits connexin-related communication pathways like gap junctions and connexin hemichannels. Keywords: ATP release, blood-brain barrier, calcium waves, connexins, cytokines. The breaching of the blood-brain barrier, i.e. the loss of interendothelial tight junctions, is an essential aspect in the pathogenesis of neuroinflammatory diseases like multiple sclerosis and AIDS dementia (Petito and Cash 1992;Poser 1993). There is now ample evidence that cytokines like tumour necrosis factor alpha (TNF-a), interleukin-1-beta (IL1-b) and interferon gamma (IFN-c) released from lymphocytes, macrophages and many other cell types, play a key role in the opening of the barrier (de Vries et al. 1996;Anthony et al. 1997;Munoz-Fernandez and Fresno 1998). These cytokines stimulate the expression of the cell adhesion molecules intercellular adhesion molecule-1 (ICAM-1) and vascular cell adhesion molecule-1 (VCAM-1) on brain endothelium, thereby facilitating lymphocyte adherence. The subsequent lymphocyte-endothelial interaction activates diverse signalling cascades: a protein kinase C (PKC) and tyrosine kinase pathway, acting at occludins and the zonula occludens-1 (ZO-1) accessory protein to disrupt tight junctions, and a Rho based pathway activating the actin cytoskeleton and tearing the tight junction proteins away from the cell-cell interface (Pfau et al. 1995; Bolton et al. Received June 17, 2003; revised manuscript received August 21, 2003; accepted September 25, 2003. Address correspondence and reprint requests to Luc Leybaert, Department Physiology and Pathophysiology, Ghent University, De Pintelaan 185 (Block B, Rm 306), B-9000 Ghent, Belgium. E-mail: Luc.Leybaert@UGent.be Abbreviations used: InsP 3 , inositol tr...

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