Simon P. Hardy

Okadaic acid‐sensitive activation of Maxi Cl− channels by triphenylethylene antioestrogens in C1300 mouse neuroblastoma cells

1 The regulation of Maxi Cl− channels by 17β‐oestradiol and non‐steroidal triphenylethylene antioestrogens represents a rapid, non‐classical effect of these compounds. In the present study we have investigated the signalling pathways used for the regulation of Maxi Cl− channel activity by oestrogens and antioestrogens in C1300 neuroblastoma cells. 2 Whole‐cell Maxi Cl− currents were readily and reversibly activated by tamoxifen, toremifene and the membrane‐impermeant ethyl‐bromide tamoxifen, only when applied to the extracellular medium. 3 Pre‐treatment of C1300 cells with oestrogen or cAMP prevented the antioestrogen‐induced activation of Maxi Cl− channels. The inhibitory effect of 17β‐oestradiol and cAMP was abolished by the kinase inhibitor staurosporine. 4 Current activation was unaffected by the removal of intracellular Ca2+ and Mg2+, but was completely abolished in the presence of okadaic acid. These results are consistent with the participation of an okadaic acid‐sensitive serine/threonine protein phosphatase in the activation of Maxi Cl− channels. However, neither oestrogen or antioestrogen treatment modified the total activity of the two major serine/threonine phosphatases, PP1 and PP2A, in C1300 cells. 5 Although the role of these Maxi Cl− channels remains unknown, our findings suggest strongly that their modulation by oestrogens and antioestrogens is linked to intracellular signalling pathways.

Cytotoxicity of the Bacillus cereus Nhe Enterotoxin Requires Specific Binding Order of Its Three Exoprotein Components

ABSTRACT This study focuses on the interaction of the three components of the Bacillus cereus Nhe enterotoxin with particular emphasis on the functional roles of NheB and NheC. The results demonstrated that both NheB and NheC were able to bind to Vero cells directly while NheA lacked this ability. It was also shown that Nhe-induced cytotoxicity required a specific binding order of the individual components whereby the presence of NheC in the priming step as well as the presence of NheA in the final incubation step was mandatory. Priming of cells with NheB alone and addition of NheA plus NheC in the second step failed to induce toxic effects. Furthermore, in solution, excess NheC inhibited binding of NheB to Vero cells, whereas priming of cells with excess NheC resulted in full toxicity if unbound NheC was removed before addition of NheB. By using mutated NheC proteins where the two cysteine residues in the predicted β-tongue were replaced with glycine (NheCcys−) or where the entire hydrophobic stretch was deleted (NheChr−), the predicted hydrophobic β-tongue of NheC was found essential for binding to cell membranes but not for interaction with NheB in solution. All data presented here are compatible with the following model. The first step in the mode of action of Nhe is associated with binding of NheC and NheB to the cell surface and probably accompanied by conformational changes. These events allow subsequent binding of NheA, leading to cell lysis.

Inhibition of ligand-gated cation-selective channels by tamoxifen

The nonsteroidal antioestrogen tamoxifen has been shown to block a number of voltage-gated cation-selective channels but its effect on ligand-gated cation-selective channels has not been studied. We have investigated the action of tamoxifen and the related derivative toremifene on ligand-gated cationic nicotinic acetylcholine and 5-HT3 receptor channels. Tamoxifen and toremifene both inhibited cationic currents of adult-type human muscle nicotinic acetylcholine receptors expressed in Xenopus oocytes with similar IC50 values of 1.2 +/- 0.03 microM (nH = 0.84 +/- 0.02) and 1.2 +/- 0.1 microM (nH = 1.1 +/- 0.1), respectively. Tamoxifen could also block native 5-HT3 receptors in NG108-15 neuroblastoma/glioma hybrid cells with IC50 = 0.81 +/- 0.15 microM and nH of 1.3 +/- 0.3. The characteristics of block by tamoxifen at the 5-HT3 receptor were voltage- and use-independent. The inhibition of the 5-HT-evoked currents were not overcome by increasing concentrations of 5-HT consistent with a noncompetitive mechanism of block.

Clostridium perfringens type A enterotoxin forms mepacrine-sensitive pores in pure phospholipid bilayers in the absence of putative receptor proteins.

Clostridium perfringens enterotoxin (CPE) is an important cause of food poisoning with no significant homology to other enterotoxins and its mechanism of action remains uncertain. Although CPE has recently been shown to complex with tight junction proteins, we have previously demonstrated that CPE increases ionic permeability in single Caco-2 cells using the whole-cell patch-clamp technique, thereby excluding any paracellular permeability. In this paper we demonstrate that CPE forms pores in synthetic phospholipid membranes in the absence of receptor proteins. The properties of the pores are consistent with CPE-induced permeability changes in Caco-2 cells suggesting that CPE has innate pore-forming ability.

Activation of Maxi Cl(-) channels by antiestrogens and phenothiazines in NIH3T3 fibroblasts.

The identification of alternative estrogen actions has been accumulating steadily over the past two decades. Typically, these novel actions are not directly related to nuclear transcriptional events but related to the interaction of estrogens with sites present at plasma membrane or cytosolic locations. These alternative effects, widely known as non-genomic effects, range from the modulation of plasma membrane ion channel activity to the regulation of different intracellular signalling cascades. In the present study we have investigated the modulation of a large conductance chloride channel (Maxi Cl(-)) by estrogens, non-steroidal triphenylethylene antiestrogens and phenothiazines in NIH3T3 fibroblasts and the dependence on guanosine triphosphate (GTP) of the Maxi Cl(-) activation. Our data identifies the non-steroidal antiestrogens toremifene and tamoxifen, and the phenothiazines chlorpromazine and triflupromazine as activators of Maxi Cl(-) channels. In contrast, 17 beta-estradiol and cAMP, added prior to the exposure to antiestrogens, prevent channel activation. The pure antiestrogen ICI 182780 did not activate the channel nor prevent its activation by non-steroidal antiestrogens. The activation of Maxi Cl(-) channels by toremifene and tamoxifen required the presence of intracellular nucleotides and was inhibited by the stable analog, GDP beta -S, suggesting the participation of a G-protein in the activation process. Little is known about the physiological relevance of Maxi Cl(-) channels. However, that fact that its regulation by estrogens and antiestrogens is shared by different cell types might imply a common role which needs to be identified.

Volume-activated chloride currents in HeLa cells are blocked by Tamoxifen but not by a membrane impermeant quaternary analogue

Background/Aims: Tamoxifen has been shown to inhibit volume activated chloride currents in many cell types. Tamoxifen has also been reported to inhibit a number of cation channels as well as cytosolic proteins such as calmodulin. The mechanism of channel block by tamoxifen is not known but three hypotheses can be proposed: i) a direct effect following binding to the channel protein from the aqueous environment or ii) a direct effect on the channel protein after partitioning into the lipid membrane or iii) an indirect mechanism via binding to intracellular regulatory proteins after diffusion across the lipid membrane. The aim of these experiments was to distinguish between these hypothoses using membrane permeant and impermeant antioestrogens. Methods: Volume activated chloride currents were recorded from single HeLa cells using whole cell patch clamp technique. The ability of tamoxifen and its membrane impermeant quaternary derivative ethyl bromide tamoxifen (EBT) to inhibit these currents was examined. Results: Extracellular tamoxifen at 3µM inhibited volume activated chloride currents in HeLa cells whereas EBT had no effect up to 10µM when applied either to the extracellular bathing solution or the intracellular solution via the patch pipette. Conclusion: Eliminating the ability of tamoxifen to cross the plasma membrane abolishes its channel blocking activity against volume activated chloride channels in HeLa cells.

Membrane impermeant antioestrogens discriminate between ligand- and voltage-gated cation channels in NG108-15 cells

Native 5-HT(3) and AChR ligand-gated cation channels can be inhibited (blocked) by the non-steroidal antioestrogen tamoxifen. However, the exact site and mechanism of inhibition by tamoxifen on these channels remain unclear. We have investigated the action of the membrane impermeant quaternary derivative, ethylbromide tamoxifen (EBT), on native ligand-gated 5-HT(3) receptor channels and voltage-gated K(+) channels in NG108-15 cells using whole cell patch clamp. Extracellular EBT inhibited whole cell cationic currents of 5-HT(3) receptors with IC(50) of 0.22+/-0.4 microM (n(H)=1.05+/-0.2). The channel block was characterised by voltage independent and use independent behaviour (similar to that of tamoxifen). EBT was unable to inhibit voltage-gated K(+) currents in NG108-15 cells. This was in contrast to the inhibition by tamoxifen which, at similar concentrations, accelerated the apparent inactivation of these outward K(+) currents. The inhibition of 5-HT(3) receptors by a membrane impermeant derivative of tamoxifen supports the view that the binding site for antioestrogens is extracellular and the inhibition is not mediated through genomic/transcriptional activity.

Analysis of Campylobacter jejuni infection in the gnotobiotic piglet and genome-wide identification of bacterial factors required for infection

To investigate how Campylobacter jejuni causes the clinical symptoms of diarrhoeal disease in humans, use of a relevant animal model is essential. Such a model should mimic the human disease closely in terms of host physiology, incubation period before onset of disease, clinical signs and a comparable outcome of disease. In this study, we used a gnotobiotic piglet model to study determinants of pathogenicity of C. jejuni. In this model, C. jejuni successfully established infection and piglets developed an increased temperature with watery diarrhoea, which was caused by a leaky epithelium and reduced bile re-absorption in the intestines. Further, we assessed the C. jejuni genes required for infection of the porcine gastrointestinal tract utilising a transposon (Tn) mutant library screen. A total of 123 genes of which Tn mutants showed attenuated piglet infection were identified. Our screen highlighted a crucial role for motility and chemotaxis, as well as central metabolism. In addition, Tn mutants of 14 genes displayed enhanced piglet infection. This study gives a unique insight into the mechanisms of C. jejuni disease in terms of host physiology and contributing bacterial factors.