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M J Wakelam

Publications and source records attributed to M J Wakelam.

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

Antigen receptors on immature, but not mature, B and T cells are coupled to cytosolic phospholipase A2 activation: expression and activation of cytosolic phospholipase A2 correlate with lymphocyte maturation.

The Ag receptors on mature B and T cells are not coupled to the activation of cytosolic phospholipase A2 (cPLA2) and arachidonic acid release. Moreover, phorbol esters such as PMA, which can activate cPLA2 via mitogen-activated protein (MAP) kinase in most cell types, also failed to induce the release of arachidonate from mature cells, suggesting that the cPLA2 pathway may not be functional in mature lymphocytes. Interestingly, Western blot analysis revealed that cPLA2, which had previously been thought to be expressed ubiquitously, is not expressed in mature B or T cells and that cytosolic phospholipase A2 expression could not be up-regulated in lymphocytes following culture with a range of cytokines most likely to be involved in an immune response such as IL-1 alpha, IL-3, or TNF-alpha. In contrast, cPLA2 was shown to be expressed and activated in thymocytes and immature B cells under conditions in which ligation of the Ag receptors led to growth arrest and/or apoptosis. Taken together, these data suggest that cPLA2 does not play a role in Ag receptor-mediated lymphocyte activation, but may be involved in the molecular mechanisms underlying lymphocyte maturation and/or self tolerance by clonal deletion.

Animals↗
PubMed1996

Vascular endothelial growth factor stimulates protein kinase C-dependent phospholipase D activity in endothelial cells.

Many tumors produce vascular endothelial growth factor (VEGF), a paracrine factor acting selectively on endothelial cells. VEGF has many effects on cultured endothelial cells and mediates angiogenesis and enhanced vascular permeability in vivo. The endothelial signal transduction pathways of VEGF represent novel targets for cancer therapy because they are readily accessible to systemically administered drugs. We have examined VEGF-stimulated signals generated in HUVEC to identify potential targets for therapeutic intervention. The transphosphatidylation reaction has been used to monitor phospholipase D (PLD) activity; total inositol phosphates have been measured after prelabeling of cells with [3H]myoinositol; and intracellular free calcium has been measured using Fura-2 fluorescence. After HUVEC-stimulation with VEGF, there is an early influx of calcium (maximal by 100 seconds) followed by activation of PLD (half maximal by 100 seconds, EC50 70 pm). The PLD activity was inhibited by reducing extracellular calcium (150 nM, 50% inhibition), exposure to 12-O-tetradecanoylphorbol 13 acetate (200 nM, 24 hours, 100% inhibition), Roche 31,8220 (10 microM, 15 minutes, 72% inhibition), or genestein (100 microM, 30 minutes, 56% inhibition), which suggests a dependence on both protein kinase C and tyrosine phosphorylation. Activation of phospholipase C-catalyzed hydrolysis of phosphatidylinositol-4,5-bisphosphate was inferred from the production of inositol phosphates, although this response was slower (half maximal by 3 minutes). The phospholipase C activity was also dependent on influx of calcium and was partially inhibited by low (150 nM) extracellular calcium. PLD may be involved in mediating a number of endothelial responses to tumor-secreted VEGF, notably cytoskeleton-dependent effects such as the cell migration involved in angiogenesis. This signal transduction pathway could represent an accessible and vulnerable target for cancer therapeutic intervention and has the novelty of being located within normal cells rather than tumor cells.

Calcium↗
PubMed1995

Wortmannin and its structural analogue demethoxyviridin inhibit stimulated phospholipase A2 activity in Swiss 3T3 cells. Wortmannin is not a specific inhibitor of phosphatidylinositol 3-kinase.

Wortmannin and its structural analogue demethoxyviridin (DMV) have been reported to be specific inhibitors of phosphatidylinositol 3-kinase activity. Here we report that these compounds are not as selective as assumed and demonstrate inhibition of bombesin-stimulated phospholipase A2 activity by both wortmannin and DMV with an IC50 (2 nM) which is slightly more potent than the inhibition of insulin-stimulated phosphatidylinositol 3,4,5-trisphosphate generation in these cells (approximately 10nM). While it has not been possible to fully block in vitro phospholipase A2 activity with wortmannin, inhibition cannot be a consequence of inhibition of PI 3-kinase activity since bombesin fails to generate 3-phosphorylated lipids in the intact cell. Therefore, while wortmannin is indeed a PI 3-kinase inhibitor, it is not as specific as previously reported, and experimental conclusions based solely on its use should be treated with caution.

3T3 Cells↗
PubMed1995

Heterologous desensitization of bombesin- and vasopressin-stimulated phospholipase D activity in Swiss 3T3 fibroblasts.

Bombesin- and vasopressin-stimulated phospholipase D (PLD) activities are rapidly desensitized in 3T3 cells, in addition both agonists are subject to heterologous desensitization. Binding studies showed that homologous desensitization was partly a result of loss of cell surface receptors, whilst heterologous desensitization was independent of receptor changes. Pretreatment with either agonist reduced subsequent GTP gamma S-stimulated PLD activity by 50% whereas a pretreatment with GTP gamma S did not attenuate the response, suggesting that the G-protein or downstream effector systems were affected by receptor activation resulting in desensitization. The desensitization of receptor-stimulated PLD activation provides support for the phospholipase functioning in a key signalling pathway.

3T3 Cells↗
PubMed1995

The roles of multiple pathways in regulating bombesin-stimulated phospholipase D activity in Swiss 3T3 fibroblasts.

The regulation of bombesin-stimulated phospholipase D (PLD) activity in Swiss 3T3 fibroblasts was examined. Increasing protein-tyrosine phosphorylation by using pervanadate to inhibit tyrosine phosphatases was found to stimulate protein kinase C (PKC)-independent [3H]phosphatidylbutanol ([3H]PtdBut) accumulation within 5 min, which continued to increase up to 30 min. The stimulation of PLD activity in response to submaximal [bombesin] could be decreased by approx. 50% by the tyrosine kinase inhibitor genistein, whereas pretreatment with genistein and the PKC inhibitor Ro-31-8220 completely abolished the generation of [3H]PtdBut in response to a maximal concentration of bombesin. The addition of guanosine 5'-[gamma-thio]triphosphate (GTP[S]) into permeabilized cells resulted in an increase in [3H]PtdBut, which was abolished by depletion of cellular ATP. The additional presence of 30 microM GTP[S] did not increase the stimulation of PLD activity by any [bombesin] tested, whereas it was synergistic with that stimulated in response to phorbol 12-myristate 13-acetate. These findings suggest that bombesin-stimulated PLD activity is indirectly regulated by G-proteins, possibly through a kinase intermediate. Furthermore, activation of protein tyrosine kinases is proposed to account for the PKC-independent arm of bombesin-stimulated PLD activity. No evidence was obtained for a form of PLD directly regulated by tyrosine phosphorylation.

3T3 Cells↗
PubMed1994

Mechanical and biochemical responses to endothelin-1 and endothelin-3 in human bronchi.

In human bronchi, contractions to endothelin-1 were unaltered by atropine (10(-5) M), indomethacin (10(-6) M), nifedipine (10(-5) M), or phosphoramidon (3.67 x 10(-5) M). Endothelin-3-evoked contractions were markedly enhanced by phosphoramidon (3.67 x 10(-5) M), unaffected by atropine (10(-5) M), and attenuated by indomethacin (10(-6) M) or nifedipine (10(-5) M). Phorbol 12,13-dibutyrate (PDB, 10(-8) M) enhanced both endothelin-1- and endothelin-3 (plus phosphoramidon)-evoked responses, an effect abolished by Ro 31-8220 (3 x 10(-8) M). Contractions to endothelin-1 or endothelin-3 alone were unaltered by staurosporine 10(-8)-3 x 10(-7) M) or Ro 31-8220 (3 x 10(-9)-3 x 10(-8) M). Endothelin-1 (3 x 10(-7) M), but not endothelin-3 (10(-10)-3 x 10(-7) M), evoked a rise in levels of inositol (1,4,5) trisphosphate (Ins(1,4,5)P3). These results suggest that endothelin-1 does not act via cyclo-oxygenase metabolites nor require Ca2+ influx via dihydropyridine-sensitive channels. It evokes Ins(1,4,5)P3 production, but does not rely upon protein kinase C activation for contraction. Endothelin-3-evoked contractions are partly mediated by cyclo-oxygenase metabolites. Endothelin-3 does not stimulate phosphatidylinositol 4,5-bisphosphate (PIP2) hydrolysis, nor utilise protein kinase C to produce contraction, but its actions may rely upon extracellular Ca2+.

Atropine↗
PubMed1994

Second messengers. Sphingolipid signalling.

Sphingomyelin metabolites seem increasingly likely to be intracellular messengers controlling a variety of cell activities, but it is not yet clear exactly which metabolites control which functions, or how they do so.

Animals↗
PubMed1994

Epidermal growth factor stimulates distinct diradylglycerol species generation in Swiss 3T3 fibroblasts: evidence for a potential phosphatidylcholine-specific phospholipase C-catalysed pathway.

Stimulation of 3T3 fibroblasts with epidermal growth factor (EGF) results in an increase in 1,2-diacylglycerol (DAG) mass which is maximal at 25 s, declining at 1 min and returning to basal levels by 30 min. No changes in alkylacylglycerol or alkenylacylglycerol were detected. Three species account for most of this mass increase: 18:0/20:5,n-3, 18:0/20:4,n-6 and 18:0/20:3,n-9. These species are characteristic of the phosphoinositides; however, previous work failed to detect any EGF-stimulated rise in inositol phosphates in these cells [Cook and Wakelam (1992) Biochem. J. 285, 247-253]. This ruled out phosphoinositide hydrolysis by phospholipase C, but raised the possibility of phospholipase D/phosphatidate phosphohydrolase-catalysed hydrolysis of phosphatidylinositol. The inclusion of butanol in the incubation medium failed to block the diacylglycerol changes, indicating that the phospholipase D pathway is not involved and that DAG must be derived from another source, probably via phospholipase C-catalysed hydrolysis of a phosphatidylcholine pool that is particularly rich in these species. The tyrosine kinase inhibitor ST-271 almost abolished the elevation in 18:0/20:5,n-3, 18:0/20:4, n-6 and 18:0/20:3,n-9 at 25 s, but only reduced the rise in total DAG mass by about 50%. The protein kinase C (PKC) inhibitor Ro-31-8220 increased DAG levels at all time points but had no effect on the species profiles. This provides additional evidence for PKC-mediated regulation of cell-surface EGF receptors, since the inhibition of PKC would increase the availability and/or ligand binding affinity of receptors at the plasma membrane and hence increase and prolong the response to EGF.

3T3 Cells↗
PubMed1994

Rapid desensitization and resensitization of bombesin-stimulated phospholipase D activity in Swiss 3T3 cells.

The kinetics of bombesin-stimulated phospholipase D (PLD) activity were examined in Swiss 3T3 fibroblasts. The stimulated activity was found to rapidly desensitize, being completely absent after 40 s. This activity then quickly, but incompletely, resensitized, with PLD being detectable after a 4.5 min wash of the desensitized cells and 75-80% of the activity being recovered after 10 min. The desensitization was dose-dependent; however, the half-maximal stimulatory concentration of bombesin was an order of magnitude lower than that required for bombesin-stimulated second messenger generation and the KD for bombesin receptor binding. This suggested that desensitization was stimulated by a 'downstream' effect, but experiments have ruled out changes in protein kinase C activity and Ca2+ concentration. Binding experiments suggested that part of the desensitization is due to receptor internalization, and the requirement for an extracellular agonist for resensitization implies that receptor recycling plays a role. Over an extended time course, cycles of desensitization and resensitization of bombesin-stimulated PLD activity were apparent which may be relevant to mitogenic signalling. These studies add further evidence for a second messenger pathway of PLD activation, and the disparity between the kinetics of diacylglycerol generation and PLD activation supports the possibility that phosphatidic acid may have a messenger role in stimulated cells.

3T3 Cells↗
PubMed1994

Angiotensin II enhances responses to endothelin-1 in bovine bronchial smooth muscle.

Angiotensin II and endothelin-1 are putative mediators in asthma. In this study we have examined the effect of angiotensin II on endothelin-1-induced contractions in bovine bronchi and the receptor types involved in the response to these agonists. Angiotensin II alone is very low in potency, producing only small contractions. In the presence of angiotensin II 10(-7) or 3 x 10(-7) M, contractions evoked by endothelin-1 were markedly enhanced. The AII1-receptor antagonist, losartan, abolished this enhancement suggesting that angiotensin II exerts this effect via an AII1-receptor. The contraction evoked by endothelin-1 is mediated via an EtA-receptor subtype since the EtA-receptor antagonist FR139317 attenuated the response. This is offset by an inhibitory EtB-type receptor, resulting in a larger contraction when these receptors are desensitized. Indeed, the EtB-receptor agonist sarafotoxin S6c reversed methacholine-evoked tone in a concentration-dependent manner. In conclusion, angiotensin II potentiates contractions evoked by endothelin-1 in bovine bronchi. This may be a mechanism by which angiotensin II--which has little activity in bronchi--may evoke substantial changes in airway tone. Angiotensin II evokes this potentiation via AII1-receptors, whilst endothelin-1 evokes contraction via EtA-receptors, an action which is offset by an inhibitory effect of EtB-receptors.

Angiotensin II↗
PubMed1994

Regulation by hypoxia of endothelin-1-stimulated phospholipase D activity in sheep pulmonary artery cultured smooth muscle cells.

1. The aim of the study was to characterize the effects of hypoxia on agonist-stimulated phospholipase D (PLD) and phospholipase C activity of sheep pulmonary artery cultured smooth muscle cells. 2. Endothelin-1 (ET-1), 5-hydroxytryptamine (5-HT) and the protein kinase C (PKC) activator tetradecanoylphorbol acetate (TPA), stimulated a time- and concentration-dependent increase in [3H]-phosphatidylbutanol accumulation. This was abolished by pretreatment of the cells with the PKC inhibitor, Ro-318220, suggesting that agonist-stimulated phospholipase D activity is dependent upon the activation of PKC. 3. Hypoxia (PO2 20 mmHg for 30 min) stimulated basal [3H]-phosphatidylbutanol accumulation by approximately 2 fold and this activity was abolished by preincubation of the cells with 10 microM Ro-318220. 4. In cells preincubated in low O2 containing medium for 30 min, the subsequent agonist-stimulated accumulation of [3H]-phosphatidylbutanol was reduced. However, the decrease in stimulation was greater for ET-1 and 5-HT than for TPA. 5. ET-1 and TPA stimulated a time-dependent increase in protein kinase C- mediated psuedosubstrate phosphorylation. Following preincubation for 30 min in low O2 containing media, basal pseudosubstrate phosphorylation increased whilst the fold stimulation by TPA and ET-1 decreased. 6. In cells preincubated in low O2 containing medium, ET-1-stimulated [3H]-inositol phosphate accumulation was reduced by approximately 30-40%. This reduction was reversed by preincubation of the cells with Ro-318220. 7. These results suggest a role for PKC in the effects of hypoxia on PLD in pulmonary artery smooth muscle cells.

Amino Acid Sequence↗
PubMed1994

Mechanical and biochemical responses to endothelin-1 and endothelin-3 in bovine bronchial smooth muscle.

1. In this study, mechanical responses to endothelin-1 and endothelin-3 were examined in bovine bronchial smooth muscle. In addition, the involvement of phosphatidylinositol 4,5-bisphosphate hydrolysis (PIP2) in the responses to these peptides was assessed by measurement of inositol (1,4,5) trisphosphate (I(1,4,5)P3) production using a specific mass assay. 2. ET-1 evoked contractions of bovine bronchi which were concentration-dependent and initiated at between 10(-9) M and 10(-8) M. ET-1-evoked responses were unaffected by slight elevation of tone with potassium chloride (3 x 10(-2) M), methacholine (10(-6) M) or U46619 (10(-7) M). 3. Contractions to ET-1 were not altered by pre-incubation with atropine (10(-5) M), indomethacin (10(-5) M), nifedipine (10(-5) M), phosphoramidon (3.67 x 10(-5) M) or by removal of the epithelium. 4. ET-3 evoked small contractions which were not concentration-dependent. In the presence of phosphoramidon (3.67 x 10(-5) M) however, concentration-dependent contractions were obtained to ET-3 which were unaffected by atropine (10(-5) M) or by removal of the epithelium, but were significantly attenuated by indomethacin (10(-5) M). Nifedipine (10(-5) M) virtually abolished this response. 5. Both ET-1 and ET-3 (in the presence of phosphoramidon)-evoked contractions were significantly enhanced by the presence of the phorbol ester phorbol 12,13-dibutyrate (10(-8) M). Neither ET-1-, nor ET-3-mediated responses were antagonized by the protein kinase C (PKC) inhibitor, Ro 31-8220 (3 x 10(-9) - 3 x 10(-8) M). 6. ET-1 (3 x 10(-7) M) evoked a biphasic rise in levels of I(1,4,5)P3 which was unaltered by preincubation with atropine, whilst ET-3 (10(-10) - 3 x 10(-7) M) failed to alter levels of I(1,4,5)P3 at any time point examined, even in the presence of phosphoramidon (3.67 x 10(-5) M). 7. These results suggest that, in bovine bronchial smooth muscle, ET-l does not evoke contraction via cyclo-oxygenase metabolites, does not evoke release of the neurotransmitter substance acetylcholine, or require calcium influx via dihydropyridine-sensitive channels. ET-1 evokes 1(1,4,5)P3 production, but stimulation of protein kinase C may not be critical for the associated contraction. In contrast,ET-3-evoked contractions are partly mediated by cyclo-oxygenase metabolites. ET-3 does not stimulate PIP2 hydrolysis, nor activate PKC, but may, either directly or as a requirement of intermediates released in response to ET-3, rely upon extracellular calcium.

Animals↗
PubMed1993

Insulin and platelet-derived growth factor acutely stimulate glucose transport in 3T3-L1 fibroblasts independently of protein kinase C.

Insulin and platelet-derived growth factor (PDGF) are mitogenic for murine 3T3-L1 fibroblasts. Both these mitogens acutely stimulate glucose transport by 2-4-fold in these cells, evident within minutes of agonist exposure. The tumour promoter and protein kinase C activator, phorbol 12-myristate 13-acetate (PMA) also stimulates glucose transport by 2-3-fold over a similar time frame, suggesting that protein kinase C may be involved in the mitogenic action of insulin and PDGF in this cell line. In an attempt to address this, we have measured intracellular sn-1,2-diacylglycerol (DAG) levels in response to insulin, PDGF and PMA. We show that PDGF and PMA induce a rapid elevation in intracellular diacylglycerol levels, but insulin was without effect. In addition, we have shown that PMA and PDGF, but not insulin, stimulate protein kinase C activity. However, depletion of protein kinase C by overnight exposure to PMA, abolished PMA-stimulated glucose transport but had no effect on insulin- and PDGF-stimulated glucose transport, suggesting that the stimulation of glucose transport by these mitogens does not involve protein kinase C. The use of the selective protein kinase C inhibitor, Roche 31-8220, which inhibited PMA-stimulated glucose transport, but was without effect on insulin- and PDGF-stimulated glucose transport further supports this conclusion. Taken together, these data argue against a role for protein kinase C in the stimulation of glucose transport in 3T3-L1 fibroblasts caused by acute exposure to insulin or PDGF.

3T3 Cells↗
PubMed1993

Bombesin stimulates distinct time-dependent changes in the sn-1,2-diradylglycerol molecular species profile from Swiss 3T3 fibroblasts as analysed by 3,5-dinitrobenzoyl derivatization and h.p.l.c. separation.

We have developed procedures for the analysis of endogenous diradylglycerol (DRG) molecular species using derivatization with 3,5-dinitrobenzoyl chloride. The introduction of this strong chromatophore enabled us to separate less than 1 nmol of DRG into its three classes (diacylglycerol, alkylacylglycerol and alkenylacylglycerol) using a combination of h.p.l.c. and t.l.c. followed by reversed-phase h.p.l.c. to resolve these classes into their component molecular species. When applied to Swiss 3T3 mouse fibroblasts stimulated with bombesin for 25 s, 5 min or 30 min, subtle time-dependent changes in the DRG patterns were observed, with only certain polyunsaturated 1,2-diacyglycerol species [18:0/20:3(n-9), 18:0/20:4(n-6), 18:0/20:4(n-3), 18:0/20:5(n-3), 18:1(n-9)/20:3(n-9), 18:1(n-9)/20:4(n-6), 16:0/22:6(n-3), 18:0/20:3(n-6) and 16:0/20:5(n-3)] showing significant agonist-stimulated increases. The amounts of the first six species were all raised at 25 s, whereas all except the latter two were elevated at 5 min. By 30 min these last species were also increased but 18:0/20:3(n-9) had returned to basal levels. Overall DRG levels, as measured by total molecular-species peak area, remained effectively constant. No changes in the amount or species profile of 1-alkyl-2-acylglycerol were observed. Comparison of these species with the acyl-chain structure of phospholipids supports the idea that inositol lipids could be the source of DRG at early stimulation times, but phosphatidylcholine appears to be a phospholipase substrate at all times. These results indicate sequential activation of several phospholipases with different substrate specificities and/or access to different phospholipid pools. They also suggest that only polyunsaturated DRGs act as second messengers and that changes in the relative amounts of these species may trigger activation of different proteins and/or isoforms (e.g. the different isoforms of protein kinase C).

3T3 Cells↗