Abstract-In resistance arteries, spread of hyperpolarization from the endothelium to the adjacent smooth muscle is suggested to be a crucial component of dilation resulting from endothelium-derived hyperpolarizing factor (EDHF). To probe the role of endothelial gap junctions in EDHF-mediated dilation, we developed a method, which was originally used to load membrane impermeant molecules into cells in culture, to load connexin (Cx)-specific inhibitory molecules rapidly (Ϸ15 minutes) into endothelial cells within isolated, pressurized mesenteric arteries of the rat. Validation was achieved by luminally loading cell-impermeant fluorescent dyes selectively into virtually all the arterial endothelial cells, without affecting either tissue morphology or function. The endothelial monolayer served as an effective barrier, preventing macromolecules from entering the underlying smooth muscle cells. Using this technique, endothelial cell loading either with antibodies to the intracellular carboxyl-terminal region of Cx40 (residues 340 to 358) or mimetic peptide for the cytoplasmic loop (Cx40; residues 130 to 140) each markedly depressed EDHF-mediated dilation. In contrast, multiple antibodies directed against different intracellular regions of Cx37 and Cx43, and mimetic peptide for the intracellular loop region of Cx37, were each without effect. Furthermore, simultaneous intra-and extraluminal incubation of pressurized arteries with inhibitory peptides targeted against extracellular regions of endothelial cell Cxs ( 43 Gap 26, 40 Gap 27, and 37,43 Gap 27; 300 mol/L each) for 2 hours also failed to modify the EDHF response. High-resolution immunohistochemistry localized Cx40 to the end of endothelial cell projections at myoendothelial gap junctions. These data directly demonstrate a critical role for Cx40 in EDHF-mediated dilation of rat mesenteric arteries. Key Words: endothelium-derived hyperpolarizing factor Ⅲ myoendothelial gap junctions Ⅲ endothelium-dependent dilation Ⅲ acetylcholine Ⅲ connexin 40 I n arterioles and some arteries, gap junctions between endothelial and smooth muscle cells (myoendothelial gap junctions [MEGJs]) enable changes in membrane potential to spread over considerable distances and, as a consequence, regulate blood flow by coordinating diameter change through the microcirculation. 1-3 For example, injection of hyperpolarizing current into a single endothelial cell can evoke extensive relaxation involving many smooth muscle cells throughout an isolated arteriole. 4,5 An important aspect of this response is that MEGJs may provide a crucial route for endothelial cell hyperpolarization to spread radially to the adjacent smooth muscle and evoke the dilation attributed to endothelium-derived hyperpolarizing factor (EDHF). 6 -8 In many arteries, there is more than 1 underlying mechanism for endothelium-dependent hyperpolarization of smooth muscle cells. In the rat mesenteric artery, during submaximal contraction to phenylephrine (PE), EDHF dilation appears to reflect hyperpolarizing current spread thro...

ATP can be released from endothelial cells, and this release is increased by intraluminal flow in blood vessels. In the present study, the effect of extracellular ATP (1 microM) on flow-induced vasodilatation was investigated in isolated and pressurized rat small mesenteric arteries. In the absence of extracellular ATP, only 46% of arteries developed dilatation in response to flow, and this response was both transient and unstable. In marked contrast, with ATP present, all vessels developed a prolonged and stable dilatation in response to flow. Even in the vessels that failed to respond to flow in the absence of ATP, dilatation could be stimulated once ATP was present. The ability of ATP to facilitate flow-induced vasodilatation was mimicked by UTP (1 microM), a P2Y agonist, or 3'-O-(4-benzoyl)benzoyl ATP (BzATP; 10 microM), an agonist for P2X1, P2X7, and P2Y11 purinoceptors. The involvement of P2X7 purinoceptors was further supported by the inhibitory effect of KN-62 (1 microM), a P2X7 antagonist, on the action of BzATP. P2X1 and P2X3 purinoceptors were not involved because their receptor agonist alpha,beta-methylene ATP had no effect. The facilitating effect of ATP on flow dilatation was also attenuated by the combined application of reactive blue 2 (100 microM), a P2Y antagonist, and suramin (100 microM), a nonselective P2X and P2Y antagonist. Furthermore, flow-induced dilatation obtained in the presence of ATP was reproducible. In contrast, in the additional presence of the ectonucleotidase inhibitor ARL-67156 (10 microM), although the first dilatation was normal, the responses to the second and later exposures to flow were greatly attenuated. The nonhydrolyzable ATP analogs adenosine-5'-(3-thiotriphosphate)trilithium salt (1 microM) and adenosine 5'-(beta,gamma-imido) triphosphate tetralithium salt hydrate (10 microM) had similar effects to those of ARL-67156. These data suggest that ATP acts through both P2X and P2Y purinoceptors to facilitate flow-induced vasodilatation and that ectonucleotidases prevent this effect by degrading ATP on the endothelial cell surface.

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Secretory Leucoprotease Inhibitor (SLPI) was originally identifed as an inhibitor of neutrophil elastase (NE). More recently, an antiinflammatory role has been described for SLPI. Oxidation of SLPI renders it inactive towards NE, however, it is not known how oxidation alters the anti-inflammatory activity of SLPI. We have investigated the effect of oxidation on the anti-inflammatory activity of SLPI and its ability to downregulate lipopolysaccharide (LPS)induced transcription factor activity in U937 cells. We have demonstrated that exogenously applied wild type SLPI can prevent activation of NFkB by LPS, an effect that is lost upon oxidation of SLPI. LPS induces the degradation of a number of NFkB regulatory proteins including IRAK, IkB alpha and IkB beta. Wild type SLPI, but not oxidised SLPI, can prevent the LPS-induced degradation of these proteins. SLPI also inhibits LPS-induced T N F alpha mRNA expression, an effect that is lost upon oxidation of SLPI. This work demonstrates that oxidation of SLPI not only reduces its capacity to function as an antiprotease but also diminishes its anti-inflammatory properties. 74The Jurkat cell line has constitutively active PI3K/PKB SIgnalling: effect of inducible expression of a membranelocalised SHIP constructThe regulation of D-3 phosphoinositides (PIS) by phosphoinositide-3 kinase (PI3K) is integral to signal transduction by key receptors that control T lymphocyte function. Jurkat cells, a lymphoblastic leukaemic cell line, are one of the most studied models of T cell signalling but have high P1(3,4,5)P3 levels due to the absence of the key lipid phosphatases PTEN and SHIP. The constitutively high levels of PI(3,4,5)P3 seen in Jurkats correlates with constitutive phosphorylation and activation of the Ser/Thr protein kinase PKB, which is critical for PI3K mediated effects on transcription factors. The absence of at least 2 endogenous lipid phosphatases makes Jurkats a good model to study the effects of transfected SHIP or PTEN either individually, or in combination. In this study, tetracycline controlled expression of the active SHIP phosphatase domain in a membrane localised chimeric protein, resulted in markedly reduced basal PI3K activity, the levels of which are markedly upregulated following CD28 stimulation. We have also compared the effects of wild type SHIP and PTEN with phosphatase inactive mutants of both proteins. The effect of expression of these proteins on membrane localisation and phosphorylation of PKB and subsequent downstream signalling events will also be discussed.NFKB is a major regulator of inflammatory genes. Its activation is tightly controlled by the inhibitor IKB, which upon stimulation becomes phosphorylated by specific kinases (IKKs), ubiquitinated and degraded. Luciferase reporter assays using the IL-8 promoter showed that both I K K a and IKKP enhanced activation of IL-8 in a concentration dependent manner. The effect was further increased by IL-I stimulation, resulting in the most prominent effect with IKKP, corresponding to 3-fold enhancem...