Search

Human lung mast cells (HLMC) express the Ca(2+)-activated K(+) channel K(Ca)3.1, which plays a crucial role in their migration to a variety of diverse chemotactic stimuli. K(Ca)3.1 activation is attenuated by the beta(2)-adrenoceptor and the adenosine A(2A) receptor through a G(s)-coupled mechanism independent of cyclic AMP. Prostaglandin E(2) promotes degranulation and migration of mouse bone marrow-derived mast cells through the G(i)-coupled EP(3) prostanoid receptor, and induces LTC(4) and cytokine secretion from human cord blood-derived mast cells. However, PGE(2) binding to the G(s)-coupled EP(2) receptor on HLMC inhibits their degranulation. We show that EP(2) receptor engagement closes K(Ca)3.1 in HLMC. The EP(2) receptor-specific agonist butaprost was more potent than PGE(2) in this respect, and the effects of both agonists were reversed by the EP(2) receptor antagonist AH6809. Butaprost markedly inhibited HLMC migration induced by chemokine-rich airway smooth muscle-conditioned media. Interestingly, PGE(2) alone was chemotactic for HLMC at high concentrations (1 microM), but was a more potent chemoattractant for HLMC following EP(2) receptor blockade. Therefore, the G(s)-coupled EP(2) receptor closes K(Ca)3.1 in HLMC and attenuates both chemokine- and PGE(2)-dependent HLMC migration. EP(2) receptor agonists with K(Ca)3.1 modulating function may be useful for the treatment of mast cell-mediated disease.

The human ether-a-go-go related gene (hERG) potassium channel is expressed in a variety of tissues including the heart, neurons and some cancer cells. hERG channels are modulated by several intracellular signalling pathways and these provide important mechanisms for regulating cellular excitability. In this study, we investigated muscarinic modulation of hERG currents and direct phosphorylation of channel subunits expressed in HEK-293 cells at physiologically relevant temperatures by protein kinase C (PKC). Activation of Gαq/11-coupled M3-muscarinic receptors with methacholine, reduced current amplitudes at all potentials with minor effects on the voltage dependence of activation and inactivation. The response to methacholine was insensitive to intracellular BAPTA, but was attenuated by either acute inhibition of PKC with 300 nm bisindolylmaleimide-1 (bis-1) or chronic down-regulation of PKC isoforms by 24 h pretreatment of cells with phorbol 12-myristate 13-acetate (PMA). Stimulation of PKC with 1-oleoyl 2-acetylglycerol (OAG), an analogue of diacylglycerol (DAG), mimicked the actions of muscarinic receptor stimulation. Direct phosphorylation of hERG was measured by [32P]orthophosphate labelling of immunoprecipitated protein with an anti-hERG antibody. Basal phosphorylation was high in unstimulated cells and further increased by OAG. The OAG dependent increase was abolished by bis-1 and down-regulation of PKC, but basal levels of phosphorylation were unchanged. Deletion of the amino-terminus of hERG prevented both the modulation of channel activity and the increase of phosphorylation by OAG. Our results are consistent with calcium and/or DAG sensitive isotypes of PKC modulating hERG currents through a mechanism that involves direct phosphorylation of sites on the amino terminus of hERG.

Highlights •Two hospitals implemented booster vaccination programmes in response to suspected staff exposure to pertussis. •Perceived vulnerability of patients was the single most important reason for healthcare workers accepting vaccination. •Pertussis vaccine uptake was high among those who had not received influenza vaccination the previous year. •Self-reported adverse effect profile following vaccination was reassuring. •Fourteen percent of staff reported a recent illness compatible with pertussis but had not notified occupational health.

A large and detailed data set is used to examine the influence of airports and airport light paths on housing prices. The results indicate that individuals consider airport proximity and airport flight patterns in their housing purchases. This shows that there exist two distinct measurable price gradients that distinguish large airports from small airports. In addition, homes located under the flight path of a large airport have a price gradient that is significantly larger than homes located under the flight path of a small airport.

Purpose: This case series describes the early radiographic and clinical results of attempted transcatheter ovarian vein (OV) embolization in 11 women with symptoms that were suggestive of the pelvic congestion syndrome (PCS). Methods: Eleven women (mean age, 33.1 years) who were multiparous were referred for lower extremity or vulvar varicosities (n = 8) or for tubo-ovarian varicosities (n = 3). After a clinical diagnosis of PCS was established, the women underwent ovarian (n = 5) or ovarian and iliac vein (n = 6) venography. Enlarged or incompetent OVs were embolized with 0.035-inch stainless steel coils or with 0.018-inch platinum microcoils and absorbable gelatin sponge. Incompetent tributaries to hypogastric veins were embolized as well (n = l). Symptoms before embolization and after embolization were recorded with a standard questionnaire, and the post-embolization symptoms were expressed as individual and overall percent relief. Results: Nine of the 11 women underwent embolization. Embolization of both OVs (n = 4), of the left OV alone (n = 4), or of a left obturator vein that communicated with vulvar varices (n = l) was performed. Eight of the 9 women (88.9%) had more than 80% immediate relief. Overall and individual symptom relief varied from 40% to 100% at the mean 13.4-month follow-up. One woman with variant anatomy and one woman with evidence of prior left OV thrombosis were not treated. There were no major complications. Two women had a mild to moderate return of the symptoms at 6 and 22 months. Conclusions: Transcatheter embolization provides excellent initial and variable midterm relief in women with typical PCS symptoms and with OV or OV and internal iliac (hypogastric) tributary vein incompetence. This interventional technique may replace or complement the traditional surgical approaches to this rarely recognized and poorly understood disease. (J Vasc Surg 1998;28:862-8.)

The human ether-a-go-go related gene (hERG) potassium channel is expressed in a variety of tissues including the heart, neurons and some cancer cells. hERG channels are modulated by several intracellular signalling pathways and these provide important mechanisms for regulating cellular excitability. In this study, we investigated muscarinic modulation of hERG currents and direct phosphorylation of channel subunits expressed in HEK-293 cells at physiologically relevant temperatures by protein kinase C (PKC). Activation of G(alpha q/11)-coupled M(3)-muscarinic receptors with methacholine, reduced current amplitudes at all potentials with minor effects on the voltage dependence of activation and inactivation. The response to methacholine was insensitive to intracellular BAPTA, but was attenuated by either acute inhibition of PKC with 300 nm bisindolylmaleimide-1 (bis-1) or chronic down-regulation of PKC isoforms by 24 h pretreatment of cells with phorbol 12-myristate 13-acetate (PMA). Stimulation of PKC with 1-oleoyl 2-acetylglycerol (OAG), an analogue of diacylglycerol (DAG), mimicked the actions of muscarinic receptor stimulation. Direct phosphorylation of hERG was measured by [(32)P]orthophosphate labelling of immunoprecipitated protein with an anti-hERG antibody. Basal phosphorylation was high in unstimulated cells and further increased by OAG. The OAG dependent increase was abolished by bis-1 and down-regulation of PKC, but basal levels of phosphorylation were unchanged. Deletion of the amino-terminus of hERG prevented both the modulation of channel activity and the increase of phosphorylation by OAG. Our results are consistent with calcium and/or DAG sensitive isotypes of PKC modulating hERG currents through a mechanism that involves direct phosphorylation of sites on the amino terminus of hERG.

The human ether-a-go-go-related gene (hERG) potassium channel is expressed in a variety of cell types, including neurons, tumor cells, and cardiac myocytes. In the heart, it is important for repolarization of the cardiac action potential. Attenuation of hERG current can cause long QT syndrome and cardiac arrhythmias such as torsades de pointes. Caffeine is frequently used as a pharmacological tool to study calcium-dependent transduction pathways in cellular preparations. It raises cytosolic calcium by opening ryanodine receptors and may also inhibit phosphodiesterases to increase cytosolic cAMP. In this study, we show 5 mM caffeine rapidly and reversibly attenuates hERG currents expressed in human embryonic kidney 293 cells to 61.1 +/- 2.2% of control. Caffeine-dependent inhibition of hERG current is not altered by raising cAMP with forskolin, buffering cytosolic calcium with 1,2-bis(2-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid, or inhibition of protein kinase C. Thus, the effects of caffeine are unlikely to be mediated by cAMP or intracellular calcium-dependent mechanisms. Further experiments showed caffeine directly blocks hERG in an open state-dependent manner. Furthermore, caffeine inhibition is greatly reduced by the pore mutants Y562A and F656A hERG, which disrupt block of most previously tested hERG antagonists. Thus, caffeine attenuates hERG currents by binding to a drug receptor located within the inner cavity of the channel. Dietary intake of caffeine is unlikely to cause long QT syndrome because plasma concentrations do not reach sufficiently high levels to significantly inhibit hERG currents. However, the effects of caffeine have implications for its use in examining calcium-dependent pathways in cellular preparations expressing hERG.

A series of six hemodialysis vascular access patients was evaluated with duplex Doppler sonography and angiography to identify Doppler sonographic parameters that are predictors of venous outflow stenosis. This preliminary series indicates that no venous outflow stenosis was present angiographically when the resistive index gradient across the venous anastomosis is less than or equal to zero and a significant venous outflow stenosis (50 to 100%) was present when the resistive index gradient is 0.15 or greater (P = 0.002, r = 0.963). Doppler acquisition of resistive index gradients across regions of venous outflow is a promising, noninvasive means of rapid radiologic evaluation of hemodialysis graft patients, especially when the need for angiography is equivocal.

Background Mast cells play a key role in asthma and recent evidence indicates that their ongoing activation in this disease is mediated, in part, via IgE in the absence of antigen. In this study we have examined whether IgE alone enhances human lung mast cell (HLMC) survival. Methods Purified HLMC were cultured for 4 weeks and survival assays then performed over 10 days following cytokine withdrawal in the presence or absence of human myeloma IgE. Quantitative real time RT-PCR was carried out to examine IL-6 mRNA expression and IL-6 protein was measured in HLMC supernatants by ELISA. Results IgE alone promoted the survival of HLMC in a dose-dependent manner following cytokine withdrawal. IgE-induced survival was eliminated with the addition of neutralising anti-IL-6 antibody but not by the addition of neutralising anti-stem cell factor. IgE sensitisation initiated profound upregulation of IL-6 mRNA in HLMC, and IL-6 concentrations were also raised in the culture supernatants of IgE-exposed cells. Conclusion These data taken together suggest that IgE in the absence of antigen promotes HLMC survival through the autocrine production of IL-6. This provides a further mechanism through which IL-6 and IgE contribute to the pathogenesis of asthma, and through which anti-IgE therapy might achieve its therapeutic effect.