Objectives: Cryopreserved blood vessels are being increasingly employed in vascular reconstruction procedures but freezing/thawing is associated with significant cell death that may lead to graft failure. Vascular cells express connexin proteins that form gap junction channels and hemichannels. Gap junction channels directly connect the cytoplasm of adjacent cells and may facilitate the passage of cell death messengers leading to bystander cell death. Two hemichannels form a gap junction channel but these channels are also present as free non-connected hemichannels. Hemichannels are normally closed but may open under stressful conditions and thereby promote cell death. We here investigated whether blocking gap junctions and hemichannels could prevent cell death after cryopreservation.Materials and methods: Inclusion of Gap27, a connexin channel inhibitory peptide, during cryopreservation and thawing of human saphenous veins and femoral arteries was evaluated by terminal deoxynucleotidyl transferase dUTP nick end labelling (TUNEL) assays and histological examination.Results: We report that Gap27 significantly reduces cell death in human femoral arteries and saphenous veins when present during cryopreservation/thawing. In particular, smooth muscle cell death was reduced by 73% in arteries and 71% in veins, while endothelial cell death was reduced by 32% in arteries and 51% in veins.Conclusions: We conclude that inhibiting connexin channels during cryopreservation strongly promotes vascular cell viability. (C) 2012 European Society for Vascular Surgery. Published by Elsevier Ltd. All rights reserved.

Connexin-43 (Cx43), a predominant cardiac connexin, forms gap junctions (GJs) that facilitate electrical cell-cell coupling and unapposed/nonjunctional hemichannels that provide a pathway for the exchange of ions and metabolites between cytoplasm and extracellular milieu. Uncontrolled opening of hemichannels in the plasma membrane may be deleterious for the myocardium and blocking hemichannels may confer cardioprotection by preventing ionic imbalance, cell swelling and loss of critical metabolites. Currently, all known hemichannel inhibitors also block GJ channels, thereby disturbing electrical cell-cell communication. Here we aimed to characterize a nonapeptide, called Gap19, derived from the cytoplasmic loop (CL) of Cx43 as a hemichannel blocker and examined its effect on hemichannel currents in cardiomyocytes and its influence in cardiac outcome after ischemia/reperfusion. We report that Gap 19 inhibits Cx43 hemichannels without blocking GJ channels or Cx40/pannexin-1 hemichannels. Hemichannel inhibition is due to the binding of Gap19 to the C-terminus (CT) thereby preventing intramolecular CT-CL interactions. The peptide inhibited Cx43 hemichannel unitary currents in both HeLa cells exogenously expressing Cx43 and acutely isolated pig ventricular cardiomyocytes. Treatment with Gap19 prevented metabolic inhibition-enhanced hemichannel openings, protected cardiomyocytes against volume overload and cell death following ischemia/reperfusion in vitro and modestly decreased the infarct size after myocardial ischemia/reperfusion in mice in vivo. We conclude that preventing Cx43 hemichannel opening with Gap19 confers limited protective effects against myocardial ischemia/reperfusion injury.

Connexin mimetic peptides corresponding to short conserved extracellular loop sequences of connexins have been used widely as reversible inhibitors of gap junctional intercellular communication. These peptides also block movement of ATP and Ca(2+) across connexin hemichannels, i.e. hexameric channels yet to dock with partners in aligned cells and to generate the gap junction cell-cell conduit. By means of electrophysiology, we compared the effects of Gap26, a mimetic peptide corresponding to a short linear sequence in the first extracellular loop of connexin43, on connexin channel function in HeLa cells expressing connexin43. We demonstrate that Gap26 inhibited electrical coupling in cell pairs mediated by gap junctions after exposure for 30min. In contrast, Gap26 applied to single cells, inhibited hemichannel currents evoked in low Ca(2+) solution with a response time of less than 5min. The results further support the view that the likely primary and direct inhibitory effect of Gap26 is on connexin hemichannels, with gap junctions becoming inhibited later. The mechanism of action of Gap26 in blocking hemichannels and gap junction channels is discussed in the context of their different functions and locations.

Gap junctions are key components underpinning multicellularity. They provide cell to cell channel pathways that enable direct intercellular communication and cellular coordination in tissues and organs. The channels are constructed of a family of connexin (Cx) membrane proteins. They oligomerize inside the cell, generating hemichannels (connexons) composed of six subunits arranged around a central channel. After transfer to the plasma membrane, arrays of Cx hemichannels (CxHcs) interact and couple with partners in neighboring attached cells to generate gap junctions. Cx channels have been studied using a range of technical approaches. Short peptides corresponding to sequences in the extra- and intracellular regions of Cxs were used first to generate epitope-specific antibodies that helped studies on the organization and functions of gap junctions. Subsequently, the peptides themselves, especially Gap26 and -27, mimetic peptides derived from each of the two extracellular loops of connexin43 (Cx43), a widely distributed Cx, have been extensively applied to block Cx channels and probe the biology of cell communication. The development of a further series of short peptides mimicking sequences in the intracellular loop, especially the extremity of the intracellular carboxyl tail of Cx43, followed. The primary inhibitory action of the peptidomimetics occurs at CxHcs located at unapposed regions of the cell's plasma membrane, followed by inhibition of cell coupling occurring across gap junctions. CxHcs respond to a range of environmental conditions by increasing their open probability. Peptidomimetics provide a way to block the actions of CxHcs with some selectivity. Furthermore, they are increasingly applied to address the pathological consequences of a range of environmental stresses that are thought to influence Cx channel operation. Cx peptidomimetics show promise as candidates in developing new therapeutic approaches for containing and reversing damage inflicted on CxHcs, especially in hypoxia and ischemia in the heart and in brain functions.

Connexin mimetic peptides (CxMPs), such as Gap26 and Gap27, are known as inhibitors of gap junction channels but evidence is accruing that these peptides also inhibit unapposed/non-junctional hemichannels (HCs) residing in the plasma membrane. We used voltage clamp studies to investigate the effect of Gap26/27 at the single channel level. Such an approach allows unequivocal identification of HC currents by their single channel conductance that is typically ~220 pS for Cx43. In HeLa cells stably transfected with Cx43 (HeLa-Cx43), Gap26/27 peptides inhibited Cx43 HC unitary currents over minutes and increased the voltage threshold for HC opening. By contrast, an elevation of intracellular calcium ([Ca(2+)](i)) to 200-500 nM potentiated the unitary HC current activity and lowered the voltage threshold for HC opening. Interestingly, Gap26/27 inhibited the Ca(2+)-potentiated HC currents and prevented lowering of the voltage threshold for HC opening. Experiments on isolated pig ventricular cardiomyocytes, which display strong endogenous Cx43 expression, demonstrated voltage-activated unitary currents with biophysical properties of Cx43 HCs that were inhibited by small interfering RNA targeting Cx43. As observed in HeLa-Cx43 cells, HC current activity in ventricular cardiomyocytes was potentiated by [Ca(2+)](i) elevation to 500 nM and was inhibited by Gap26/27. Our results indicate that under pathological conditions, when [Ca(2+)](i) is elevated, Cx43 HC opening is promoted in cardiomyocytes and CxMPs counteract this effect.

Gap junctions (GJ) are essential conduits that underpin cell-to-cell coupling between cardiomyocytes and are formed from the coalescence of connexin (Cx) hemichannels from two close opposed cells. However, there is evidence supporting a (patho)physiologic role for 'unpaired' Cx hemichannels as they traffic through the plasma membrane (PM) to the GJ. We used beating HL-1 cardiomyocyte monolayers as a model system for GJ intercellular communication (GJIC) and to investigate the functional role of Cx hemichannels in syncitial behaviour.

The cytoplasmic Ca2+ concentration ([Ca2+]i) is an important factor determining the functional state of blood-brain barrier (BBB) endothelial cells but little is known on the effect of dynamic [Ca2+]i changes on BBB function. We applied different agonists that trigger [Ca2+]i oscillations and determined the involvement of connexin channels and subsequent effects on endothelial permeability in immortalized and primary brain endothelial cells. The inflammatory peptide bradykinin (BK) triggered [Ca2+]i oscillations and increased endothelial permeability. The latter was prevented by buffering [Ca2+]i with BAPTA, indicating that [Ca2+]i oscillations are crucial in the permeability changes. Bradykinin-triggered [Ca2+]i oscillations were inhibited by interfering with connexin channels, making use of carbenoxolone, Gap27, a peptide blocker of connexin channels, and Cx37/43 knockdown. Gap27 inhibition of the oscillations was rapid (within minutes) and work with connexin hemichannel-permeable dyes indicated hemichannel opening and purinergic signaling in response to stimulation with BK. Moreover, Gap27 inhibited the BK-triggered endothelial permeability increase in in vitro and in vivo experiments. By contrast, [Ca2+]i oscillations provoked by exposure to adenosine 5’ triphosphate (ATP) were not affected by carbenoxolone or Gap27 and ATP did not disturb endothelial permeability. We conclude that interfering with endothelial connexin hemichannels is a novel approach to limiting BBB-permeability alterations.

As expression of Cxs in cells of the immune system increases upon cellular activation, we investigated whether Cxs and especially CxHcs play a major role during T cell-mediated responses. In particular, we studied the expression of Cx43Hc following CD4(+) T cell stimulation using flow cytometry, real-time PCR, and Western blot analysis. We showed that expression of Cx43 and its phosphorylated isoforms increased in response to the engagement of CD3 and CD28. Cx43Hcs were found to be involved in sustaining proliferation of T cells, as assessed by cell cycle staining, thymidine incorporation assays, and CFSE analysis of cells exposed to mimetic peptide inhibitors of the plasma membrane Cx channels and antibodies generated to an extracellular region of Cx. The reduction of T cell proliferation mediated by Cx channel inhibitors suppressed cysteine uptake but not cytokine production. We conclude that upon antigen recognition, T cells require CxHc to sustain their clonal expansion. J. Leukoc. Biol. 88: 79-86; 2010.

The increasing use of nanoparticles in medicine has raised concerns over their ability to gain access to privileged sites in the body. Here, we show that cobalt-chromium nanoparticles (29.5 +/- 6.3 nm in diameter) can damage human fibroblast cells across an intact cellular barrier without having to cross the barrier. The damage is mediated by a novel mechanism involving transmission of purine nucleotides (such as ATP) and intercellular signalling within the barrier through connexin gap junctions or hemichannels and pannexin channels. The outcome, which includes DNA damage without significant cell death, is different from that observed in cells subjected to direct exposure to nanoparticles. Our results suggest the importance of indirect effects when evaluating the safety of nanoparticles. The potential damage to tissues located behind cellular barriers needs to be considered when using nanoparticles for targeting diseased states.

Human bone marrow is a clinical source of autologous progenitor stem cells showing promise for cardiac repair following ischemic insult. Functional improvements following delivery of adult bone marrow CD34(+) cells into heart tissue may require metabolic/electrical communication between participating cells. Since connexin43 (Cx43) channels are implicated in cardiogenesis and provide intercellular connectivity in the heart, the authors analyzed the expression of 20 connexins (Cx) in CD34(+) cells and in monocytes and granulocytes in bone marrow and spinal cord. Reverse transcriptase-polymerase chain reaction (RT-PCR) detected only low expression of Cx43 and Cx37. Very low level dye coupling was detected by flow cytometry between CD34(+) cells and other Cx43 expressing cells, including HL-1 cardiac cells, and was not inhibited by specific gap junction inhibitors. The results indicate that CD34(+) cells are unlikely to communicate via gap junctions and the authors conclude that use of CD34(+) cells to repair damaged hearts is unlikely to involve gap junctions. The results concur with the hypothesis that bone marrow cells elicit improved cardiac function through release of undefined paracrine mediators.

Connexin hemichannels have a low open probability under normal conditions but open in response to various stimuli, forming a release pathway for small paracrine messengers. We investigated hemichannel-mediated ATP responses triggered by changes of intracellular Ca(2+) ([Ca(2+)](i)) in Cx43 expressing glioma cells and primary glial cells. The involvement of hemichannels was confirmed with gja1 gene-silencing and exclusion of other release mechanisms. Hemichannel responses were triggered when [Ca(2+)](i) was in the 500nM range but the responses disappeared with larger [Ca(2+)](i) transients. Ca(2+)-triggered responses induced by A23187 and glutamate activated a signaling cascade that involved calmodulin (CaM), CaM-dependent kinase II, p38 mitogen activated kinase, phospholipase A2, arachidonic acid (AA), lipoxygenases, cyclo-oxygenases, reactive oxygen species, nitric oxide and depolarization. Hemichannel responses were also triggered by activation of CaM with a Ca(2+)-like peptide or exogenous application of AA, and the cascade was furthermore operational in primary glial cells isolated from rat cortex. In addition, several positive feed-back loops contributed to amplify the responses. We conclude that an elevation of [Ca(2+)](i) triggers hemichannel opening, not by a direct action of Ca(2+) on hemichannels but via multiple intermediate signaling steps that are adjoined by distinct signaling mechanisms activated by high [Ca(2+)](i) and acting to restrain cellular ATP loss.

Many cell signalling pathways are driven by changes in cytosolic calcium. We studied the effects of a range of inhibitors of connexin channels on calcium signalling in cardiac cells and HeLa cells expressing connexins. Gap 26 and 27, peptides that mimic short sequences in each of the extracellular loops of connexin 43, and anti-peptide antibodies generated to extracellular loop sequences of connexins, inhibited calcium oscillations in neonatal cardiac myocytes, as well as calcium transients induced by ATP in HL-1 cells originating from cardiac atrium and HeLa cells expressing connexin 43 or 26. Comparison of single with confluent cells showed that intracellular calcium responses were suppressed by interaction of connexin mimetic peptides and antibodies with hemichannels present on unapposed regions of the plasma membrane. To investigate how inhibition of hemichannels in the plasma membrane by the applied reagents was communicated to calcium store operation in the endoplasmic reticulum, we studied the effect of Gap 26 on calcium entry into cells and on intracellular IP3 release; both were inhibited by Gap 26. Calcium transients in both connexin 43- and connexin 26-expressing HeLa cells were inhibited by the peptides suggesting that the extended cytoplasmic carboxyl tail domain of larger connexins and their interactions with intracellular scaffolding/auxiliary proteins were unlikely to feature in transmitting peptide-induced perturbations at hemichannels in the plasma membrane to IP3 receptor channel central to calcium signalling. The results suggest that calcium levels in a microenvironment functionally connecting plasma membrane connexin hemichannels to downstream IP3-dependent calcium release channels in the endoplasmic reticulum were disrupted by the connexin mimetic peptide, although implication of other candidate hemichannels cannot be entirely discounted. Since calcium signalling is fundamental to the maintenance of cellular homeostasis, connexin hemichannels emerge as therapeutic targets open to manipulation by reagents interacting with external regions of these channels.

We studied the role of connexin hemichannels in the release of ATP by neonatal cardiac myocytes subject to ischaemic stress. Mechanical, osmotic and oxidative stress and changes in extracellular or intracellular Ca2+ levels induce connexin hemichannels located in the plasma membrane to open and release small ions and molecules with signaling potential such as ATP. Since ATP release has been implicated in adaptation to oxygen deprivation, we studied its release by cardiac myocytes incubated in a custom-built hypoxia chamber for various periods. In a simulated ischaemia model (0.5% oxygen and 0.2 g/l glucose) a peak of ATP release occurred at 80 min followed by a return to steady state levels for a further 200 min. This peak of ATP release was not observed in myocytes subject to hypoxia (0.5% oxygen, 3.0 g/l glucose). ATP release in ischaemia was influenced by two classes of reagents that target connexins, the channel forming proteins of gap junctions. First, the connexin hemichannel inhibitors Gap 26 and 18a glycyrrhetinic acid abolished the ATP peak of release. Second, the AAP10, a peptide with antiarrhythmic properties markedly increased the peak of ATP release observed at 80 min of ischaemia and also induced a second smaller peak at 180-240 min. ATP content of the myocytes and Cx43 phosphorylation were monitored. Since the release of ATP in ischaemia was abolished by connexin channel inhibitors and stimulated by a peptide developed to target connexins in the context of cardiac arrhythmia, the results suggest that nucleotide release by connexin hemichannels is likely to feature in the response of myocytes to ischaemic stress in the heart. (C) 2008 Elsevier B.V. All rights reserved.

Gap junctions (GJs) have been demonstrated to communicate cell death signals from apoptotic to healthy cells, thereby spatially extending apoptosis. Before being incorporated into GJs, hemichannels (hemi-GJs) are normally closed but recent evidence suggests that they can be opened by various messengers and conditions, thereby forming a pore through which molecules can enter or leave the cell potentially leading to cell death. The aim of this study was to determine the contribution of GJs and hemichannels in the communication of apoptosis toward surrounding cells. We induced apoptosis in C6 glioma cells stably transfected with connexin (Cx)43, with cytochrome C (cytC) using in situ electroporation and found that healthy surrounding cells underwent apoptotic transformation. Work with various cell death markers, wild-type (WT) and Cx43-expressing cells, inhibitors of GJs and/or hemichannels, and Cx43 gene silencing showed that GJs contribute to the spread of apoptosis in a zone next to where apoptosis was triggered whereas hemichannels also promoted cell death beyond this area. Buffering cytoplasmic Ca(2+) changes inhibited the spread of apoptosis in both cases. We conclude that Cx43 hemichannels, in concert with their GJ counterparts, play a role in communicating cytC-induced apoptotic cell death messages.

Gap junction channels constructed of connexins (Cxs) are expressed by peripheral and secondary lymphoid organ-derived lymphocytes. These channels in the plasma membrane play key roles in a range of lymphocyte functions exemplified by the synthesis and secretion of Igs and cytokines and during transmigration across the endothelium. Most recently, their involvement in antigen cross-presentation has also been established. We report here for the first time the expression of mRNA and protein encoding Cx43 in mouse-derived CD4(+) Th0, Th1, and Th2 lymphocyte subpopulations and demonstrate the establishment gap junction channel formation with primary macrophages in vitro. We show that this mode of direct communication is particularly favored in Th1-macrophage interactions and that LPS inhibits lymphocyte-macrophage cross-talk independently of the subset of lymphocyte involved. Our work suggests that gap junction-mediated communication can be modulated in the absence of specific antigenic stimulation. Therefore, a further mechanism featuring gap junction-mediated communication may be implicated in immune regulation.

There is a dearth of chemical inhibitors of connexin-mediated intercellular communication. The advent of short "designer" connexin mimetic peptides has provided new tools to inhibit connexin channels quickly and reversibly. This perspective describes the development of mimetic peptides, especially Gap 26 and 27 that are the most popular and correspond to specific sequences in the extracellular loops of connexins 37, 40 and 43. Initially they were used to inhibit gap-junctional coupling in a wide range of mammalian cells and tissues. Currently, they are also being examined as therapeutic agents that accelerate wound healing and in the early treatment of spinal cord injury. The mimetic peptides bind to connexin hemichannels, influencing channel properties as shown by lowering of electrical conductivity and potently blocking the entry of small reporter dyes and the release of ATP by cells. A mechanism is proposed to help explain the dual action of connexin mimetic peptides on connexin hemichannels and gap-junctional coupling.