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E R Chilvers

Publications and source records attributed to E R Chilvers.

At least 55 records · Page 3Linked to original sources
PubMed1996

A regulatory role for cAMP in phosphatidylinositol 3-kinase/p70 ribosomal S6 kinase-mediated DNA synthesis in platelet-derived-growth-factor-stimulated bovine airway smooth-muscle cells.

In bovine airway smooth-muscle cells platelet-derived growth factor (PDGF) and endothelin (Et-1) stimulate sustained and comparable activation of mitogen-activated protein kinase (MAP kinase) but display very different mitogenic efficacies, with PDGF inducing 50 times more DNA synthesis than Et-1. To examine additional signalling pathways which may be involved in this response, we investigated the role of phosphatidylinositol 3-kinase (PtdIns 3-kinase)/p70 ribosomal protein S6 kinase (p70s6k) in mediating PDGF- and Et-1-induced mitogenesis, and whether inhibition of this pathway may underly the ability of cAMP to inhibit cell proliferation. PDGF stimulated an increase in PtdIns 3-kinase activity and a sustained 15-fold increase in p70s6k activity that was abolished by both wortmannin and rapamycin. Et-1, however, stimulated only a 2-fold increase in p70s6k activity that was rapamycin-sensitive but wortmannin-insensitive. DNA synthesis stimulated by PDGF (50-fold) and Et-1 (2-fold) followed a similar pattern of inhibition. Pretreatment with phorbol ester did not affect p70s6k activation in response to PDGF. Raising intracellular cAMP levels using forskolin, however, resulted in a marked time-dependent inhibition of p70s6k activity, a decrease in the tyrosine phosphorylation of the PtdIns 3-kinase p85 subunit and reduced PtdIns 3-kinase activity. Forskolin also inhibited PDGF-stimulated DNA synthesis. These results suggest that PtdIns 3-kinase-dependent activation of p70s6k may determine mitogenic efficacy of agonists that generate comparable MAP kinase signals. Negative regulation of PtdIns 3-kinase by cAMP may play an important role in the inhibition of airway smooth-muscle cell proliferation.

Androstadienes↗
PubMed1996

Inhibition of neuropeptide-stimulated tyrosine phosphorylation and tyrosine kinase activity stimulates apoptosis in small cell lung cancer cells.

Small cell lung cancer (SCLC) cell growth is sustained by multiple autocrine and paracrine growth loops involving neuropeptides. The bombesin family of peptides are autocrine growth factors in H345 SCLC cells and provide a paradigm for the study of growth factors and mitogenic signaling in SCLC cells. We show that bombesin (and other neuropeptides) stimulates protein tyrosine phosphorylation (particularly focal adhesion kinase) and protein tyrosine kinase (PTK) activity in intact SCLC cells. Furthermore, the broad spectrum neuropeptide receptor antagonist [D-Arg, D = Phe, D-Trp, Leu11]substance P inhibits all neuropeptide-mediated signals (including PTK activation), SCLC cell growth in vivo and in vitro, and also increases the natural rate of apoptosis seen in growing SCLC cell lines. Hence the effect of selective PTK inhibition on SCLC cell growth and apoptosis was examined. We show that selective inhibition of PTK activity, with genistein and (3,4,5-tri-hydroxyphenyl)-methylene(-propanedinitrile) tyrphostin-25 inhibits basal and neuropeptide-stimulated SCLC cell growth. Genistein and tyrphostin-25 also stimulate apoptosis in SCLC cells. Inhibition of proliferation in these cells is intimately linke to apoptosis, because these changes occurred without any effect on SCLC cell cycle kinetics, suggesting that apoptosis occurs independently of the cell cycle and that failure to progress through the cell cycle results in apoptosis. Because tyrphostin-25 fails to influence p53 or Bcl-2 expression in these cells, this mode of programmed cell death appears to be via a p53- and Bcl-2-independent mechanism. These results provide evidence that tyrosine phosphorylation is a mitogenic signal in SCLC cells and suggest that regulation of the level of protein tyrosine phosphorylation represents a critical determinant of whether SCLC cells survive and proliferate or die by apoptosis. Thus PTK inhibition may provide a novel therapeutic option in SCLC that has become resistant to conventional chemotherapeutic agents.

Apoptosis↗
PubMed1996

Neuropeptides stimulate tyrosine phosphorylation and tyrosine kinase activity in small cell lung cancer cell lines.

Stimulation of small cell lung cancer (SCLC) cells with neuropeptides bombesin, bradykinin, gastrin, and neurotensin resulted in increased tyrosine kinase activity and tyrosine phosphorylation of a number of polypeptides including a p120 kDa polypeptide identified by immunoblotting as focal adhesion kinase (p125FAK). The neuropeptides stimulated a rapid, concentration-dependent phosphorylation of p125FAK (EC50 of 1 nM, 5 nM, and 2 nM for bombesin, bradykinin, and gastrin, respectively), which was receptor mediated and inhibited by both specific and broad-spectrum neuropeptide receptor antagonists. Specific inhibition of protein tyrosine kinase activity by tyrphostin-25 inhibited both basal and neuropeptide-stimulated SCLC cell growth. These results identify a novel neuropeptide-stimulated growth signaling event in SCLC cells.

Bombesin↗
PubMed1996

Priming differentially regulates neutrophil adhesion molecule expression/function.

Lung injury in a variety of disease states is critically dependent on neutrophil-mediated inflammatory responses. Neutrophil recruitment to sites of infection or tissue damage requires co-ordinated regulation of neutrophil adhesion and activation status. We have examined the effects of treatment of human peripheral blood neutrophils with priming agents [lipopolysaccharide (LPS). tumor necrosis factor-alpha (TNF-alpha) and platelet-activating factor (PAF)] upon expression of CD11a. CD11b. CD11c. CD35 and CD62-1 and CD11b function to assess whether subtle regulation of neutrophil adhesion potential accompanies augmented formyl-methionyl-leucyl-phenylalanine-stimulated superoxide production. We have found that there are differential effects of priming concentrations of these agents. For LPS. CD62L loss occurs in the absence of changes in CD11b, whereas for PAF. CD11b up-regulation occurs in the absence of detectable loss of CD62-L. However, for TNF-2, decreased expression of CD62-L occurs concomitantly with increased expression of CD11b. In addition, we have shown that priming agents augment CD11b functional activity in a manner that parallels the priming of the respiratory burst. Thus, priming agents may differentially regulate neutrophil adhesive capacity and data presented in this manuscript suggest that the increased effector cell function observed in primed cells may be associated with a distinct repertoire of potential adhesive interactions.

Antigens, CD↗
PubMed1996

Characterization of inositol hexakisphosphate (InsP6)-mediated priming in human neutrophils: lack of extracellular [3H]-InsP6 receptors.

1. Inositol hexakisphosphate (InsP6) is a ubiquitous and abundant cytosolic inositol phosphate that has been reported to prime human neutrophils for enhanced agonist-stimulated superoxide anion generation. This led to the proposal that the release of InsP6 from necrotic cells may augment the functional responsiveness of neutrophils at an inflammatory focus. The aim of this study was to examine whether the functional effects of InsP6 in neutrophils are receptor-mediated and establish the magnitude of this priming effect relative to other better characterized priming agents. 2. Analysis of [3H]-InsP6 binding to human neutrophil membranes in 20 mM Tris, 20 mM NaCl, 100 mM KCl, 5 mM EDTA (pH 7.7) buffer using 0.1 mg ml-1 membrane protein and 2.5 nM [3H]-InsP6 (90 min, 4 degrees C), demonstrated specific low affinity [3H]-InsP6 binding that was non-saturable up to a radioligand concentration of 10 nM. 3. [3H]-InsP6 displacement by InsP6 gave a Hill coefficient of 0.55 and best fitted a two-site logistic model (53% KD 150 nM, 47% KD 5 microM). [3H]-InsP6 binding also displayed low (3 fold) selectivity for InsP6 over Ins(1,3,4,5,6)P5. 4. The specific [3H]-InsP6 binding displayed a pH optimum of 8, was abolished by pre-boiling the membranes, and was enhanced by Ca2+, Mg2+ and Na+. 5. In incubations with intact neutrophils, where high levels of specific [3H]-LTB4 binding was observed, no [3H]-InsP6 binding could be identified. 6. Preincubation of neutrophils with 100 microM InsP6 had no effect on resting cell morphology, but caused a minor and transient (maximal at 30 s) enhancement of (0.1 nM) fMLP-induced shape change (% cells shape changed: fMLP 53 +/- 3%, fMLP+InsP6 66 +/- 4%). Similarly, InsP6 (100 microM, 30 s) had no effect on basal superoxide anion generation and, compared to lipopolysaccharide (LPS, 100 ng ml-1, 60 min), tumour necrosis factor-alpha (TNF alpha, 200 u ml-1, 30 min) or platelet-activating factor (PAF, 100 nM, 5 min) caused only a small enhancement of 100 nM fMLP-stimulated superoxide anion generation (fold-increase in superoxide anion generation over fMLP alone: InsP6 1.8 +/- 0.3, LPS 6.8 +/- 0.6, TNF alpha 5.2 +/- 0.7, PAF 5.8 +/- 0.6). 7. While these data support the presence of a specific, albeit low affinity, [3H]-InsP6 binding site in human neutrophil membrane preparations, the lack of binding to intact cells implies that the functional effects of InsP6 (ie. enhanced fMLP-stimulated superoxide anion generation and shape change) are not receptor-mediated.

Adult↗
PubMed1995

Agents that elevate cAMP inhibit human neutrophil apoptosis.

Neutrophil apoptosis, determined after 20 h in culture using standard criteria and shedding of cell surface CD16 (Fc gamma RIII), is dramatically inhibited, in a concentration-dependent manner, by the cAMP analogs, dibutyryl-cAMP and 8-Br-cAMP, and the adenylyl cyclase activator, forskolin. Furthermore, the stable receptor-directed PGD2 mimetic, ZK 118.182, and the PGE2 mimetic, 11-deoxy PGE1, similarly inhibited apoptosis. The DP-receptor antagonist BW A868C blocked the effect of ZK 118.182 and the protein kinase A inhibitor H-89 reversed the inhibition of apoptosis induced by dibutyryl-cAMP. These results clearly show that neutrophil apoptosis is markedly attenuated by cAMP elevating agents. This nucleotide second messenger may play a fundamental role in controlling neutrophil longevity and pharmacological regulation of cAMP levels or actions may influence neutrophil apoptosis in vivo.

Alprostadil↗
PubMed1995

Hypoxia prolongs neutrophil survival in vitro.

Neutrophil apoptosis represents a major mechanism involved in the resolution of inflammation. Since hypoxia induces apoptosis in several cell lines and is of particular relevance in many disease states, we studied the effect of oxygen concentration on neutrophil survival in vitro. Hypoxia caused a dramatic decrease in neutrophil apoptosis (% apoptosis 20 h: 78.7 +/- 2.2% in 21% O2, 61.4 +/- 6.5% in 2.5% O2, 23.1 +/- 3.2% in 0% O2, n = 5). This was additive to the effect of GM-CSF (50 U/ml), not associated with induction of bcl-2 expression, and was not mimicked by methionine (5 mM), superoxide dismutase (200 micrograms/ml) or Trolox (10 mM) but was mimicked by catalase (250 micrograms/ml). Hence, hypoxia has a bcl-2-independent effect on neutrophil apoptosis that may adversely affect the clearance of these cells from an inflammatory focus.

Cell Hypoxia↗
PubMed1995

Lack of effect of recombinant platelet-derived growth factor on human neutrophil function.

Platelet-derived growth factor (PDGF) has been reported to induce chemotaxis, degranulation, and superoxide anion generation, and to increase the expression of CD11b/CD18 in human neutrophils; hence, it has been proposed as an important regulator of neutrophil function. Most of the studies on PDGF, however, have been complicated by the use of nonrecombinant PDGF or the use of mixed leukocyte cell preparations. Assessment of the effects of recombinant human PDGF-AB or -BB which display agonist activity against both PDGF receptor subtypes failed to demonstrate any effect of this peptide on neutrophil shape change, respiratory burst activity, CD11/CD18, or CD62-L expression, inositol 1,4,5-trisphosphate accumulation, or phosphorylation of mitogen-activated protein kinase. This apparent lack of effect of PDGF was consistent with our findings that neutrophils display no specific 125I-PDGF-AB or -BB binding and lack detectable mRNA for PDGF alpha-receptor and beta-receptors. These data indicate that human neutrophils do not possess functional PDGF receptors and question previous reports of a functional effect of this peptide in these cells.

3T3 Cells↗
PubMed1995

Stimulation by endothelin-1 of mitogen-activated protein kinases and DNA synthesis in bovine tracheal smooth muscle cells.

1. In cultures of bovine tracheal smooth muscle cells, platelet-derived growth factor-BB (PDGF), bradykinin (BK) and endothelin-1 (ET-1) stimulated the tyrosine phosphorylation and activation of both pp42 and pp44 kDa forms of mitogen-activated protein (MAP) kinase. 2. Both ET-1 and PDGF stimulated a sustained activation of MAP kinase whilst the response to BK was transient. 3. Activation of MAP kinase occurred in a concentration-dependent manner (EC50 values: ET-1, 2.3 +/- 1.3 nM; BK, 8.7 +/- 4.1 nM, PDGF, 9.7 +/- 3.2 ng ml-1). 4. Pretreatment with the protein kinase C (PKC) inhibitor Ro-318220, significantly reduced ET-1 activation of MAP kinase at 2 and 5 min but enhanced MAP kinase activation at 60 min. 5. Following chronic phorbol ester pretreatment, BK-stimulated activation of MAP kinase was abolished whilst the responses to PDGF and ET-1 were only partly reduced (80 and 45% inhibition respectively). 6. Pretreatment with pertussis toxin reduced ET-1 stimulated activation of MAP kinase particularly at later times (60 min), but left the responses to both PDGF and BK unaffected. 7. ET-1 also stimulated a 3 fold increase in [3H]-thymidine incorporation which was abolished by pertussis toxin pretreatment. In contrast, PDGF stimulated a 131 fold increase in [3H]-thymidine incorporation which was not affected by pertussis toxin. 8. These results suggest that a pertussis toxin-sensitive activation of MAP kinase may play an important role in ET-1-stimulated DNA synthesis but that activation of MAP kinase alone is not sufficient to induce the magnitude of DNA synthesis observed in response to PDGF.

Animals↗
PubMed1995

A comparison of the effects of SCA40, NS 004 and NS 1619 on large conductance Ca(2+)-activated K+ channels in bovine tracheal smooth muscle cells in culture.

1. The effects of imidazopyrazine derivative, SCA40, on the activity of single large conductance, Ca(2+)-activated K+ (BKCa) channels in inside-out and outside-out patches from bovine tracheal smooth muscle (BTSM) cells in culture have been compared with those of two established BKCa channel openers, NS 004 and NS 1619. 2. The presence of BKCa channels on inside-out patches of BTSM membranes was confirmed by the single channel conductance (240 pS), selectivity for K+, dependence of channel activity on [Ca2+]i, and sensitivity to the selective BKCa channel blocker, iberiotoxin. 3. NS 004 and ND 1619 (3-30 microM) induced concentration-related increases in open state probability of BKCa channels when applied to either inside-out or outside-out BTSM patches, thus confirming that these compounds are activators of the BKCa channel in this preparation. 4. SCA40 (0.1-10 microM) had no effect on the activity of BKCa channels when applied to either inside-out or outside-out patches which subsequently responded to the application of NS 004 (10-20 microM). 5. It is concluded that SCA40 does not have a direct effect on BKCa channel activity in BTSM patches and that the previously reported relaxant action of SCA40 on tracheal smooth muscle is unlikely to be mediated by this mechanism.

Animals↗
PubMed1994

Effects of membrane depolarization and changes in intra- and extracellular calcium concentration on phosphoinositide hydrolysis in bovine tracheal smooth muscle.

Agonist-stimulated phosphoinositide metabolism plays a central role in pharmacomechanical coupling in airways smooth muscle (ASM). In many other tissues and cells, most noteably excitable cells, membrane depolarization or an increase in intracellular Ca2+ ([Ca2+]i) generated by inositol 1,4,5-trisphosphate (Ins(1,4,5)P3)-induced Ca2+ release or agonist-mediated Ca2+ influx is able to trigger or augment phosphatidylinositol 4,5-bisphosphate (PtdIns(4,5)P2) hydrolysis and/or initiate PtdIns4P/PtdIns hydrolysis by direct stimulation of PIC. To assess the importance of these mechanisms in ASM the effects of KCl-induced membrane depolarization, extracellular Ca2+ ([Ca2+]e) chelation, and addition of ionomycin to elevate [Ca2+]i on basal and agonist-stimulated Ins(1,4,5)P3 concentration and [3H]-InsPx accumulation have been examined. Reducing [Ca2+]e from 1.8 mM to 6 or 0.8 microM caused a progressive inhibition of agonist-stimulated [3H]inositol polyphosphate accumulation over 30 min with the histamine-stimulated response being significantly more sensitive to [Ca2+]e chelation than the response to carbachol. In contrast, the initial accumulation of Ins(1,4,5)P3 was completely unaffected by such reductions in [Ca2+]e. Incubation of [3H]inositol-prelabelled BTSM slices with buffer containing 80 mM KCl failed to stimulate [3H]InsPx accumulation, causing instead a small inhibition of carbachol-stimulated [3H]InsPx accumulation with a similar effect seen with respect to Ins(1,4,5)P3 accumulation. Addition of 5 microM ionomycin to BTSM slices similarly did not stimulate Ins(1,4,5)P3 generation and only increased [3H]InsPx accumulation after prolonged stimulation in the presence of high (mM) [Ca2+]e. These data indicated that in ASM, membrane depolarization or physiological increases in [Ca2+]i did not result in either independent activation of PIC or augmentation of initial agonist-stimulated PtdIns(4,5)P2 hydrolysis. However, while the initial agonist-stimulated generation of Ins(1,4,5)P3 was not dependent on [Ca2+]e, a normal plasmalemmal Ca2+ gradient was required to sustain maximal rates of agonist-stimulated PtdIns(4,5)P2 hydrolysis.

Animals↗
PubMed1994

Effect of temperature on muscarinic cholinoceptor-mediated phosphoinositide metabolism and tension generation in bovine tracheal smooth muscle.

The effect of decreased temperature on phosphoinositide metabolism was studied in flurbiprofen pretreated bovine tracheal smooth muscle (BTSM) by investigating the consequences of cooling on muscarinic-cholinoceptor-mediated [3H]inositol phosphate ([3H]InsP) and inositol 1,4,5-trisphosphate (Ins(1,4,5)P3) accumulation, basal phosphoinositidase C (PIC) activity and airways smooth muscle (ASM) tone. Cooling of [3H]Ins labelled BTSM slices from 37 degrees C to 27 degrees C for 20 min prior to the addition of agonist caused a substantial (73.0 +/- 2.5%) inhibition of carbachol (100 microM, 30 min)-stimulated [3H]InsP accumulation compared to values measured at 37 degrees C. The degree of inhibition of [3H]InsP accumulation was similar at all agonist time points (2-30 min) studied. In parallel experiments, cooling of unlabelled BTSM slices from 37 degrees C to 27 degrees C resulted in a 34% reduction in basal Ins(1,4,5)P3 mass (37 degrees C, 13.1 +/- 0.6 pmol mg-1 protein; 27 degrees C, 8.9 +/- 0.9 pmol mg-1 protein; P < 0.02) and markedly attenuated carbachol (100 microM)-stimulated increases in Ins(1,4,5)P3 accumulation. Basal PIC activity in the soluble fraction of BTSM homogenates, measured using a [3H]phosphatidylinositol 4,5-bisphosphate (PtdIns(4,5)P2) /deoxycholate assay system, was also significantly lower at 27 degrees C compared to 37 degrees C (initial velocities of PtdIns(4,5)P2 hydrolysis of 853 +/- 167 (37 degrees C) and 418 +/- 119 (27 degrees C) pmol min-1 ml-1 (1/400 diluted) BTSM cytosol; p < 0.02).(ABSTRACT TRUNCATED AT 250 WORDS)

(4-(m-Chlorophenylcarbamoyloxy)-2-butynyl)trimethy↗
PubMed1994

Miliary tuberculosis in Edinburgh--a comparison between 1984-1992 and 1954-1967.

We have reviewed the records of 29 patients notified with miliary tuberculosis in Edinburgh from 1984 to 1992 and compared our findings with those for the 40 patients previously reported in Edinburgh from 1954 to 1967. The incidence of miliary tuberculosis has not changed. Respiratory symptoms of cough and dyspnoea were commoner in 1984-1992 (P < 0.001) perhaps reflecting the increase in mean age at presentation (73.5 vs. 59.4 years; P < 0.001). Mortality was 50% in 1984-1992, significantly higher (P < 0.05) than the 25% recorded in 1954-1967. Forty percent of cases in both time periods were of cryptic miliary disease. The diagnosis of cryptic disease tended to be made more often post-mortem and less often by a trial of anti-tuberculosis chemotherapy in 1984-1992. Our findings emphasize the current poor outcome associated with a diagnosis of miliary tuberculosis. It is important to consider this diagnosis in elderly patients with unexplained pyrexia and implement a trial of specific anti-tuberculosis chemotherapy to confirm it.

Age Distribution↗