The Connexin Mimetic Peptide Gap27 and Cx43-Knockdown Reveal Differential Roles for Connexin43 in Wound Closure Events in Skin Model Systems.

In the epidermis, remodelling of Connexin43 is a key event in wound closure. However, controversy between the role of connexin channel and non-channel functions exist. We compared the impact of SiRNA targeted to Connexin43 and the connexin mimetic peptide Gap27 on scrape wound closure rates and hemichannel signalling in adult keratinocytes (AK) and fibroblasts sourced from juvenile foreskin (JFF), human neonatal fibroblasts (HNDF) and adult dermal tissue (ADF). The impact of these agents, following 24 h exposure, on GJA1 (encoding Connexin43), Ki67 and TGF-ß1 gene expression, and Connexin43 and pSmad3 protein expression levels, were examined by qPCR and Western Blot respectively. In all cell types Gap27 (100-100 µM) attenuated hemichannel activity. In AK and JFF cells, Gap27 (100 nM-100 µM) enhanced scrape wound closure rates by ~50% but did not influence movement in HNDF or ADF cells. In both JF and AK cells, exposure to Gap27 for 24 h reduced the level of Cx43 protein expression but did not affect the level in ADF and HNDF cells. Connexin43-SiRNA enhanced scrape wound closure in all the cell types under investigation. In HDNF and ADF, Connexin43-SiRNA enhanced cell proliferation rates, with enhanced proliferation also observed following exposure of HDNF to Gap27. By contrast, in JFF and AK cells no changes in proliferation occurred. In JFF cells, Connexin43-SiRNA enhanced TGF-ß1 levels and in JFF and ADF cells both Connexin43-SiRNA and Gap27 enhanced pSmad3 protein expression levels. We conclude that Connexin43 signalling plays an important role in cell migration in keratinocytes and foreskin derived fibroblasts, however, different pathways are evoked and in dermal derived adult and neonatal fibroblasts, inhibition of Connexin43 signalling plays a more significant role in regulating cell proliferation than cell migration.

Connexins and skin disease: insights into the role of beta connexins in skin homeostasis.

Cell-to-cell communication triggered by connexin channels plays a central role in maintaining epidermal homeostasis. Here, we discuss the role of the beta connexin subgroup, where site-specific mutations in at least 4 of these proteins lead to distinctive non-inflammatory and inflammatory hyperproliferative epidermal disorders. Recent advances in the molecular pathways evoked and correlation with clinical outcome are discussed. The latest data provide increasing evidence that connexins in the epidermis are sensors to environmental stress and that targeting aberrant hemichannel activity holds significant therapeutic potential for inflammatory skin disorders.

Cell motility in models of wounded human skin is improved by Gap27 despite raised glucose, insulin and IGFBP-5.

Reducing Cx43 expression stimulates skin wound healing. This is mimicked in models when Cx43 function is blocked by the connexin mimetic peptide Gap27. IGF-I also stimulates wound healing with IGFBP-5 attenuating its actions. Further, the IGF-I to IGFBP-5 ratio is altered in diabetic skin, where wound closure is impaired. We investigated whether Gap27 remains effective in augmenting scrape-wound closure in human skin wound models simulating diabetes-induced changes, using culture conditions with raised glucose, insulin and IGFBP-5. Gap27 increased scrape-wound closure in normal glucose and insulin (NGI) and to a lesser extent in high glucose and insulin (HGI). IGF-I enhanced scrape-wound closure in keratinocytes whereas IGFBP-5 inhibited this response. Gap27 overcame the inhibitory effects of IGFBP-5 on IGF-I activity. Connexin-mediated communication (CMC) was reduced in HGI, despite raised Cx43, and Gap27 significantly decreased CMC in NGI and HGI. IGF-I and IGFBP-5 did not affect CMC. IGF-I increased keratinocyte proliferation in NGI, and Gap27 increased proliferation in NGI to a greater extent than in HGI. We conclude that IGF-I and Gap27 stimulate scrape-wound closure by independent mechanisms with Gap27 inhibiting Cx43 function. Gap27 can enhance wound closure in diabetic conditions, irrespective of the IGF-I:IGFBP-5 balance.

The connexin mimetic peptide Gap27 increases human dermal fibroblast migration in hyperglycemic and hyperinsulinemic conditions in vitro.

Significant increases in skin wound healing rates occur by reducing connexin-mediated communication (CMC). Gap27, a connexin (Cx) mimetic peptide targeted to the second extracellular loop of Cx43, which inhibits CMC, increases migration of human keratinocytes and dermal fibroblasts. To examine the efficacy of Gap27 in a hyperglycemic and hyperinsulinemic in vitro environment, cell migration, gap junction, and Cx hemichannel functionality and cell-substrate adhesion assays were performed on human dermal fibroblasts and diabetic fibroblast and keratinocytes. To investigate fibroblast genes involved in these processes, extra-cellular matrix (ECM) and adhesion gene expression was determined with a PCR array. Gap27 increased fibroblast migration in both euglycemia/euinsulinemia and hyperglycemia/hyperinsulinemia, and influenced migration in diabetic keratinocytes. Hyperglycemia/hyperinsulinemia reduced gap junction coupling in fibroblasts and Gap27 reduced CMC and cell adhesion to substrata in fibroblasts cultured in high glucose. Migrating dermal fibroblast ECM and cell adhesion genes were found to be differentially regulated by Gap27 in euglycemia and hyperglycemia. The PCR array showed that Gap27 upregulated 34 genes and downregulated 1 gene in euglycemic migrating fibroblasts. By contrast in hyperglycemia, Gap27 upregulated 1 gene and downregulated 9 genes. In euglycemic conditions, Gap27 induced upregulation of genes associated with ECM remodeling, whereas in hyperglycemia, ECM component genes were downregulated by Gap27. Thus, Gap27 improves cell migration during scrape-wound repair in hyperglycemia/hyperinsulinemia conditions in vitro, although migration of diabetic cells is less influenced. Our results suggest that this increase in motility may occur by decreasing gap junction and hemichannel activity and altering gene expression in the adhesion and ECM pathway.

Connexin 43 mimetic peptide Gap27 reveals potential differences in the role of Cx43 in wound repair between diabetic and non-diabetic cells.

During early wound healing (WH) events Connexin 43 (Cx43) is down-regulated at wound margins. In chronic wound margins, including diabetic wounds, Cx43 expression is enhanced suggesting that down-regulation is important for WH. We previously reported that the Cx43 mimetic peptide Gap27 blocks Cx43 mediated intercellular communication and promotes skin cell migration of infant cells in vitro. In the present work we further investigated the molecular mechanism of Gap27 action and its therapeutic potential to improve WH in skin tissue and diabetic and non-diabetic cells. Ex vivo skin, organotypic models and human keratinocytes/fibroblasts of young and old donors and of diabetic and non-diabetic origin were used to assess the impact of Gap27 on cell migration, proliferation, Cx43 expression, localization, phosphorylation and hemichannel function. Exposure of ex vivo WH models to Gap27 decreased dye spread, accelerated WH and elevated cell proliferation. In non-diabetic cell cultures Gap27 decreased dye uptake through Cx hemichannels and after scratch wounding cells showed enhanced migration and proliferation. Cells of diabetic origin were less susceptible to Gap27 during early passages. In late passages these cells showed responses comparable to non-diabetic cells. The cause of the discrepancy between diabetic and non-diabetic cells correlated with decreased Cx hemichannel activity in diabetic cells but excluded differences in Cx43 expression, localization and Ser368-phosphorylation. These data emphasize the importance of Cx43 in WH and support the concept that Gap27 could be a beneficial therapeutic to accelerate normal WH. However, its use in diabetic WH may be restricted and our results highlight differences in the role of Cx43 in skin cells of different origin.

Connexin mimetic peptides improve cell migration rates of human epidermal keratinocytes and dermal fibroblasts in vitro.

Nonhealing cutaneous wounds, a major cause of morbidity and mortality, are difficult to treat. Recent studies suggest that significant increases in skin wound-healing rates occur by altering gap junction intercellular communication (GJIC). As migration of keratinocytes and fibroblasts is an important feature of wound healing, this study investigated whether migration rates in cultured normal human epidermal keratinocytes and dermal fibroblasts could be altered by modulating GJIC via connexin mimetic peptides. First, HeLa cells stably transfected with connexin43 (Cx43), Cx40, or Cx26 were used as a model to determine connexin specificity and the doses of connexin mimetic peptides required to attenuate GJIC. Gap26 and Gap26M inhibited GJIC dose dependently and were nonconnexin specific, whereas Gap27 was Cx43-selective. Skin keratinocytes and fibroblasts expressed a variety of connexins, with Cx43 predominating. Cx43 protein expression was reduced at leading edges 3 hours after scraping confluent monolayers, resolving at 24 hours. Gap26M and Gap27 significantly increased migration rates across scrapes in keratinocytes and fibroblasts by blocking gap junction functionality. GJIC inhibition can thus directly influence keratinocyte and fibroblast migration. Furthermore, our results support the therapeutic potential of connexin mimetic peptides to aid wound closure, and provide a simple approach to screening new agents.

The anti-arrhythmic peptide AAP10 remodels Cx43 and Cx40 expression and function.

The anti-arrhythmic peptide AAP10 has previously been shown to acutely upregulate electrical cell-to-cell coupling mediated via connexin 43 gap junctions. In the present work, we have further examined the connexin (Cx) specificity and mechanism of action of this peptide in HeLa cells expressing Cx43, Cx40 or Cx26. The ability of cells to transfer the small fluorescent dyes Alexa 488 (MW 570) or Alexa 594 (MW 759), as markers for metabolic coupling mediated via gap junctions, before and after exposure to AAP10 and/or the protein kinase C inhibitor chelerythrine for 5 h was determined by microinjection analysis. Immunofluorescence analysis assessed the effect of AAP10 on the spatial localisation of each Cx sub-type. Cell extracts were isolated for Western blot and reverse transcription polymerase chain reaction analysis at 0, 5, 10, 18 and 24 h following exposure to AAP10 and the relative Cx expression profiles determined. AAP10 enhanced the ability of Cx43 and, to a lesser extent, Cx40 to transfer Alexa 488. It also enhanced the ability of Cx43 to transfer Alexa 594 but not Cx40. Inhibition of protein kinase C blocked this enhanced response in both Cx sub-types. Western blot analysis determined that AAP10 induced Cx40 protein expression over periods of up to 24 h with an associated increase in the localisation of Cx40 at points of cell-to-cell contact following 24-h exposure. Cx43 expression was transiently induced following exposure to the peptide for 5-10 h, with an associated increase in Cx43 at points of cell-to-cell contact, returning to control levels by 18-24 h, via a post-translational mechanism independent of chelerythrine. A transient increase in Cx40 mRNA expression but not Cx43 mRNA expression was also observed. By contrast, AAP10 had no effect on the ability of Cx26 gap junctions to transfer the dyes or on the level of Cx26 expression. We propose that AAP10 is a versatile peptide that remodels metabolic coupling via Cx43 and to a lesser extent Cx40 gap junction channels via an initial protein-kinase-C-dependent pathway modifying local responses at the plasma membrane. This is followed by enhanced Cx43 or Cx40 protein expression.

ATP release by cardiac myocytes in a simulated ischaemia model: inhibition by a connexin mimetic and enhancement by an antiarrhythmic peptide.

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 Ca(2+) 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.

The antiarrhythmic peptide rotigaptide (ZP123) increases gap junction intercellular communication in cardiac myocytes and HeLa cells expressing connexin 43.

We investigated the effects of rotigaptide (ZP123), a stable hexapeptide with antiarrhythmic properties, on gap junction mediated intercellular communication in contracting rat neonatal cardiac myocytes, HL-1 cells derived from cardiac atrium and in HeLa cells transfected with cDNA encoding Cx43-GFP, Cx32-GFP, Cx26-GFP, wild-type Cx43 or wild-type Cx26. Intercellular communication was monitored before and after treatment with rotigaptide following microinjection of small fluorescent dyes (MW<1 kDa). The communication-modifying effect of rotigaptide was confined to cells expressing Cx43 since the peptide had no effect on dye transfer in HeLa cells expressing Cx32-GFP, Cx26-GFP or wild-type Cx26. In contrast, HeLa cells expressing Cx43-GFP exposed to 50 nM rotigaptide for 5 h showed a 40% increase in gap junction mediated communication. Rotigaptide (50 nM) increased intercellular dye transfer in myocytes and atrial HL-1 cells, where Cx43 is the dominant connexin. However, it caused no change in cell beating rates of cardiac myocytes. Western blot analysis showed that rotigaptide did not modify the overall level of Cx43 expression and changes in the phosphorylation status of the protein were not observed.We conclude that the effects of rotigaptide were confined to cells expressing Cx43.

Elevated endothelial nitric oxide bioactivity and resistance to angiotensin-dependent hypertension in 12/15-lipoxygenase knockout mice.

12/15-Lipoxygenase (12/15-LOX) plays a pathogenic role in atherosclerosis. To characterize whether 12/15-LOX also contributes to endothelial dysfunction and hypertension, regulation of vessel tone and angiotensin II (ang II) responses were characterized in mice deficient in 12/15-LOX. There was a twofold increase in the magnitude of l-nitroarginine-methyl ester-inhibitable, acetylcholine-dependent relaxation or phenylephrine-dependent constriction in aortic rings isolated from 12/15-LOX(-/-) mice. Plasma NO metabolites and aortic endothelial NO synthase (eNOS) expression were also elevated twofold. Angiotensin II failed to vasoconstrict 12/15-LOX(-/-) aortic rings in the absence of L-nitroarginine-methyl ester, and ang II impaired acetylcholine-induced relaxation in wild-type, but not 12/15-LOX(-/-) rings. In vivo, 12/15-LOX(-/-) mice had similar basal systolic blood pressure measurements to wild type, however, blood pressure elevations in response to ang II infusion (1.1 mg/kg/day) were significantly attenuated (maximal pressure, 143.4 +/- 4 mmHg versus 122.1 +/- 5.3 mmHg for wild type and 12/15-LOX(-/-), respectively). In contrast, vascular hypertrophic responses to ang II, and ang II type 1 receptor (AT1-R) expression were similar in both strains. This study shows that 12/15-LOX(-/-) mice have increased NO biosynthesis and impaired ang II-dependent vascular responses in vitro and in vivo, suggesting that 12/15-LOX signaling contributes to impaired NO bioactivity in vascular disease in vivo.

Effects of connexin-mimetic peptides on gap junction functionality and connexin expression in cultured vascular cells.

1. We have investigated the effects of connexin-mimetic peptides homologous to the Gap 26 and Gap 27 domains of Cxs 37, 40 and 43 against gap junctional communication and connexin expression in rat aortic endothelial cells (RAECs) and A7r5 myocytes. 2. Immunostaining and Western blot analysis confirmed the presence of gap junction plaques containing Cx43, but not Cx40, in RAECs, whereas plaques containing Cxs 40 and 43 were evident in A7r5 cells. Expression of Cx37 was limited in RAECs and absent from A7r5 cells. 3. Under control conditions calcein-loaded RAECs transferred dye to approximately 70% of subjacent A7r5 cells after coculture for 4-5 h. Dye transfer was inhibited by a peptide targeted to Cxs 37 and 43 ((37,43)Gap 27), but minimally affected by peptides targeted to Cxs 37 and 40 ((37,40)Gap 26 and (40)Gap 27). These findings suggest that the myoendothelial gap junctions that couple RAECs and A7r5 cells are constructed principally from Cx43. 4. Inhibition of dye transfer from RAECs to A7r5 cells cocultured in the presence of (37,43)Gap 27 plus (37,40)Gap 26 for 5 h was fully reversible. 5. In A7r5 cells, endogenous expression of Cx40 and Cx43 was unaffected by incubation with (37,43)Gap 27, (37,40)Gap 26, either individually or in combination, and the peptide combination did not impair connexin trafficking or the de novo formation of gap plaques in A7r5 cells transfected to express Cx43-GFP. 6. Treatment of A7r5 cells with (37,43)Gap 27 plus (37,40)Gap 26 abolished synchronized oscillations in intracellular [Ca2+] induced by the alpha1-adrenoceptor agonist phenylephrine. 7. The reversibility and lack of effect of the peptides on plaque formation suggests that they may be considered ideal probes for functional studies of connexin-mediated communication in the vascular wall.

Incorporation of connexins into plasma membranes and gap junctions.

Gap junctions are polymeric assemblies of aligned pairs of interacting hexameric connexon hemichannel units facilitating direct intercellular communication. The principal process leading to assembly of gap junctions involves the cotranslational insertion of connexin (Cx) proteins into the endoplasmic reticulum, followed by their rapid oligomeric association into homo- or heteromeric connexons that are trafficked via the Golgi apparatus to the plasma membrane. Oligomerisation is a high-fidelity process that determines connexon channel stoichiometry and conductance characteristics. A large number of mutations in Cx26 and Cx32 detected in genetic diseases have emphasised the requirement for precise oligomerisation of connexins into hexameric connexons that traffic to the plasma membrane. Mutations in Cx43 are rare, and in the cardiovascular system, where it is the dominant connexin, disease changes are linked to its abundance and to gap junction remodelling. Connexins with short carboxyl tails may also be post-translationally inserted as oligomeric channels directly into plasma membranes. This mechanism of channel assembly is highly dependent on microtubule integrity and may allow cells to rapidly modulate gap junctional cross talk.

Ouabain exerts biphasic effects on connexin functionality and expression in vascular smooth muscle cells.

1. We have compared the effects of ouabain on the maintenance of gap junctional communication in rat aortic A7r5 smooth muscle cells, monkey COS-1 fibroblasts and human HeLa epithelial cells. 2. Ouabain (1 mM) interrupted dye coupling between confluent A7r5 cells within approximately 1 h, and high concentrations of ouabain were similarly required to reduce coupling between COS-1 cells selected to express the rat alpha1 Na+/K+-ATPase subunit, which is ouabain resistant. By contrast, low concentrations of ouabain (1-10 microM) attenuated dye transfer in wild-type COS-1 and HeLa cells, whose endogenous alpha1 subunits possess relatively high affinity for the glycoside (Ki approximately 0.3 vs approximately 100 microM) Ouabain-induced reductions in dye transfer therefore correlated with the ability of the glycoside to bind to the Na+/K+-ATPase isoenzymes expressed in these different cell lines. 3. No consistent relationship between inhibition of intercellular dye transfer and secondary changes in [Ca2+]i or pHi could be identified following incubation with ouabain. 4. In separate experiments, the effects of ouabain on real-time trafficking of connexin (Cx) protein were monitored by time-lapse microscopy of A7r5 cells transfected to express a fluorescent Cx43-green fluorescent protein (GFP) and the ability of the glycoside to modulate endogenous expression of Cx40 and Cx43 evaluated in A7r5 cells by immunochemical and Western blot analysis. 5. Ouabain (1 mM) depressed vesicular trafficking of Cx43-GFP after approximately 1 h, and caused a time-dependent loss of endogenous Cx40 and Cx43 protein that was first evident at 2 h and almost complete after 4 h. These effects of ouabain on Cx expression were reversed 90 min following washout of the glycoside. 6. We conclude that ouabain exerts biphasic effects on intercellular communication that involve an initial decrease in gap junctional permeability followed by a global reduction in the expression of Cx protein. Further studies are necessary to establish to what extent these actions of ouabain reflect inversion of the normal [Na+]i/[K+]i ratio and/or conversion of the Na+/K+-ATPase into a general signal transducer that regulates downstream protein synthesis.

Connexin channels, connexin mimetic peptides and ATP release.

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.

Ouabain exerts biphasic effects on connexin functionality and expression in vascular smooth muscle cells.

1. We have compared the effects of ouabain on the maintenance of gap junctional communication in rat aortic A7r5 smooth muscle cells, monkey COS-1 fibroblasts and human HeLa epithelial cells. 2. Ouabain (1 mM) interrupted dye coupling between confluent A7r5 cells within approximately 1 h, and high concentrations of ouabain were similarly required to reduce coupling between COS-1 cells selected to express the rat alpha1 Na+/K+-ATPase subunit, which is ouabain resistant. By contrast, low concentrations of ouabain (1-10 microM) attenuated dye transfer in wild-type COS-1 and HeLa cells, whose endogenous alpha1 subunits possess relatively high affinity for the glycoside (Ki approximately 0.3 vs approximately 100 microM) Ouabain-induced reductions in dye transfer therefore correlated with the ability of the glycoside to bind to the Na+/K+-ATPase isoenzymes expressed in these different cell lines. 3. No consistent relationship between inhibition of intercellular dye transfer and secondary changes in [Ca2+]i or pHi could be identified following incubation with ouabain. 4. In separate experiments, the effects of ouabain on real-time trafficking of connexin protein were monitored by time-lapse microscopy of A7r5 cells transfected to express a fluorescent Cx43-green fluorescent protein (GFP) and the ability of the glycoside to modulate endogenous expression of connexins (Cx) 40 and 43 evaluated in A7r5 cells by immunochemical and Western blot analysis. 5. Ouabain (1 mM) depressed vesicular trafficking of Cx43-GFP after approximately 1 h, and caused a time-dependent loss of endogenous Cx40 and Cx43 protein that was first evident at 2 h and almost complete after 4 h. These effects of ouabain on Cx expression were reversed approximately 90 min following washout of the glycoside. 6. We conclude that ouabain exerts biphasic effects on the intercellular communication that involve an initial decrease in gap junctional permeability followed by a global reduction in the expression of Cx protein. Further studies are necessary to establish to what extent these actions of ouabain reflect inversion of the normal [Na+]i/[K+]i ratio and/or conversion of the Na+/K+-ATPase into a general signal transducer that regulates downstream protein synthesis.

Pharmacological sensitivity of ATP release triggered by photoliberation of inositol-1,4,5-trisphosphate and zero extracellular calcium in brain endothelial cells.

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.

Photoliberating inositol-1,4,5-trisphosphate triggers ATP release that is blocked by the connexin mimetic peptide gap 26.

Calcium signals can be communicated between cells by the diffusion of a second messenger through gap junction channels or by the release of an extracellular purinergic messenger. We investigated the contribution of these two pathways in endothelial cell lines by photoliberating InsP(3) or calcium from intracellular caged precursors, and recording either the resulting intercellular calcium wave or else the released ATP with a luciferin/luciferase assay. Photoliberating InsP(3) in a single cell within a confluent culture triggered an intercellular calcium wave, which was inhibited by the gap junction blocker alpha-glycyrrhetinic acid (alpha-GA), the connexin mimetic peptide gap 26, the purinergic inhibitors suramin, PPADS and apyrase and by purinergic receptor desensitisation. InsP(3)-triggered calcium waves were able to cross 20 microm wide cell-free zones. Photoliberating InsP(3) triggered ATP release that was blocked by buffering intracellular calcium with BAPTA and by applying gap 26. Gap 26, however, did not inhibit the gap junctional coupling between the cells as measured by fluorescence recovery after photobleaching. Photoliberating calcium did not trigger intercellular calcium waves or ATP release. We conclude that InsP(3)-triggered ATP release through connexin hemichannels contributes to the intercellular propagation of calcium signals.

Lighting up gap junction channels in a flash.

Gap junction intercellular communication channels permit the exchange of small regulatory molecules and ions between neighbouring cells and coordinate cellular activity in diverse tissue and organ systems. These channels have short half-lives and complex assembly and degradation pathways. Much of the recent work elucidating gap junction biogenesis has featured the use of connexins (Cx), the constituent proteins of gap junctions, tagged with reporter proteins such as Green Fluorescent Protein (GFP) and has illuminated the dynamics of channel assembly in live cells by high-resolution time-lapse microscopy. With some studies, however, there are potential short-comings associated with the GFP chimeric protein technologies. A recent report by Gaietta et al., has highlighted the use of recombinant proteins with tetracysteine tags attached to the carboxyl terminus of Cx43, which differentially labels 'old' and 'new' connexins thus opening up new avenues for studying temporal and spatial localisation of proteins and in situ trafficking events.

Gap junctions: structure and function (Review).

Gap junctions are plasma membrane spatial microdomains constructed of assemblies of channel proteins called connexins in vertebrates and innexins in invertebrates. The channels provide direct intercellular communication pathways allowing rapid exchange of ions and metabolites up to approximately 1 kD in size. Approximately 20 connexins are identified in the human or mouse genome, and orthologues are increasingly characterized in other vertebrates. Most cell types express multiple connexin isoforms, making likely the construction of a spectrum of heteromeric hemichannels and heterotypic gap junctions that could provide a structural basis for the charge and size selectivity of these intercellular channels. The precise nature of the potential signalling information traversing junctions in physiologically defined situations remains elusive, but extensive progress has been made in elucidating how connexins are assembled into gap junctions. Also, participation of gap junction hemichannels in the propagation of calcium waves via an extracellular purinergic pathway is emerging. Connexin mutations have been identified in a number of genetically inherited channel communication-opathies. These are detected in connexin 32 in Charcot Marie Tooth-X linked disease, in connexins 26 and 30 in deafness and skin diseases, and in connexins 46 and 50 in hereditary cataracts. Biochemical approaches indicate that many of the mutated connexins are mistargeted to gap junctions and/or fail to oligomerize correctly into hemichannels. Genetic ablation approaches are helping to map out a connexin code and point to specific connexins being required for cell growth and differentiation as well as underwriting basic intercellular communication.

Relative contributions of NO and gap junctional communication to endothelium-dependent relaxations of rabbit resistance arteries vary with vessel size.

Two synthetic peptide inhibitors of gap junctional communication have been used to compare the contribution of direct cell-cell coupling to acetylcholine-induced relaxations of the rabbit central ear artery (G(0)) and its second branch generation (G(2)). These peptides, designated (43)Gap 26 and (37,43)Gap 27, possess sequence homology with specific domains of the first extracellular loop of connexin 43 (Cx43) and second extracellular loop of Cxs 37 and 43, respectively. Immunohistochemistry confirmed the presence of Cxs 37, 40, and 43 in the vascular endothelium, but of only Cx43 in the media of G(0). At concentrations of 300 microM, (43)Gap 26 and (37,43)Gap 27 each inhibited the maximum response to acetylcholine in G(2) by approximately 50%, but by only approximately 20% in G(0), whereas inhibition of NO synthesis by 300 microM N(G)-nitro-L-arginine methyl ester attenuated maximum relaxations to acetylcholine by approximately 30% in G(2), but by approximately 70% in G(0). Residual endothelium-derived hyperpolanizing factor-type responses in G(0) and G(2) were abolished by (43)Gap 26 and (37,43)Gap 27. In HeLa cells transfected to express a chimeric Cx43-green fluorescent protein that forms functional gap junctions, the peptides were equally effective inhibitors of Lucifer yellow dye transfer. We conclude that the contribution of gap junctions to endothelium-dependent relaxation is inversely related to vessel size and exhibits an apparently reciprocal relationship with NO-mediated mechanisms of vasorelaxation in the rabbit ear.