Biomedical subjects

W Howard Evans

Publications and source records attributed to W Howard Evans.

8 recordsLinked to original sources

PubMed2003

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.

Adenosine Triphosphate↗

PubMed2002

Post-translational integration and oligomerization of connexin 26 in plasma membranes and evidence of formation of membrane pores: implications for the assembly of gap junctions.

Gap-junction channels provide a widespread intercellular signalling mechanism. They are constructed of a family of connexin membrane proteins that thread across the membrane four times and oligomerize to generate hexameric gap-junction hemichannels. Using an in vitro cell-free transcription/translation system, we demonstrate that connexin (Cx) 26, one of the smallest connexins, is integrated directly in a post-translational manner into plasma membranes. Protein-cleavage studies of Cx26 integrated into plasma membranes indicate a similar native transmembrane topography to that of Cx26 integrated co-translationally into microsomes. Cx26 integrated post-translationally into plasma membranes oligomerizes and, when incorporated into liposomes, provides permeability to ascorbic acid, suggesting that gap-junction hemichannels are generated. The results provide the basis of a novel alternative mechanism for spontaneous assembly in plasma membranes of Cx26 gap-junction hemichannels that occurs independently of the conventional biogenesis of gap junctions involving connexin trafficking and oligomerization via membrane components of the secretory pathway.

Animals↗

PubMed2002

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.

Animals↗

PubMed2002

Gap junction intercellular communication during lymphocyte transendothelial migration.

Migration of lymphocytes across the endothelium of central or peripheral tissues, a process occurring following activation or differentiation, involves cell to cell interactions featuring adhesion and heterotypic signalling 'cross-talk'. Since lymphocytes and endothelial cells express connexins, the subunit proteins of gap junction intercellular channels, we investigated whether these channels feature in heterotypic signalling during transendothelial migration of lymphocytes. We show, using FACS analysis, that calcein, a gap junction permeant fluorescent dye, was transferred from endothelial cell layers to lymphocytes. The gap junction involvement in intercellular dye transfer was reinforced by studies showing that the process was inhibited by connexin mimetic peptides, a new class of reagents shown to block gap junction communication. Further evidence for the involvement of lymphocyte gap junctions in intercellular communication during transendothelial migration was obtained by two-photon laser scanning microscopy. Although gap junctional communication was inhibited by connexin mimetic peptides, they had little influence on the transmigration process.

Amino Acid Sequence↗

PubMed2002

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.

Acetylcholine↗

PubMed2002

Epidermal stem cells do not communicate through gap junctions.

Although enrichment of putative epidermal stem cells has been achieved, a need for additional markers that can enable isolation of live keratinocytes is crucial for characterization of these cells. Earlier work has shown that connexin proteins are absent from basal cells in the limbal epithelium, a region of the corneal epithelium enriched in corneal stem cells. Accordingly, we investigated whether connexin 43, a gap junction protein present in the basal layer of normal human epidermis, can serve as a negative marker for keratinocyte stem cells. In humans, cells with immunohistochemically undetectable levels of connexin 43 are found in the epidermal basal layer of neonatal foreskin and in the follicular bulge region. About 10% of the basal keratinocytes are connexin 43 negative, as determined by flow cytometry. These cells are uniformly small and low in granularity. Restricted gap junction communication was confirmed by the failure of low molecular weight dyes to transfer between cells. Experiments carried out in mouse epidermis demonstrated that most of the slowly cycling cells, detected as label-retaining cells, do not express connexin 43. Thus, presumptive keratinocyte stem cells can be identified and separated based on connexin 43 expression.

Adult↗

PubMed2002

Gap junctions and connexins: potential contributors to the immunological synapse.

Gap junctional communication is a widespread mechanism for metabolic coupling of adjoining cells. In the immune system, evidence has built up showing that lymphocytes possess the protein building blocks of gap junctions, the connexins. The most widespread is connexin 43, but connexin 40 is also present in secondary lymphoid organs. Inhibitors of gap junctional communication, especially the highly specific connexin mimetic peptides, have been shown to decrease the secretion of immunoglobulins and cytokines by T and B lymphocyte cocultures, indicating that connexins may play a fundamental role in lymphocyte physiology. Traditionally, connexins function when assembled into gap junction-intercellular channels. However, the possibility is now arising that gap junction hemichannels, previously viewed as plasma membrane precursors of gap junctions, are also involved in the release from cells of small metabolites, e.g., adenosine 5'-triphosphate and nicotinamide adenine dinucleotide(+), and this opens up a second, possible paracrine function for connexins detected in lymphocytes. The increasing structural and functional evidence points to a potential role that lymphocyte gap junctional intercellular communication may play within the complex signaling components of the immunological synapse.

Animals↗

PubMedDate not supplied

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.

Animals↗