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D J Withers

Publications and source records attributed to D J Withers.

24 records · Page 2Linked to original sources
PubMed1996

Dissociation of mitogen-activated protein kinase activation from p125 focal adhesion kinase tyrosine phosphorylation in Swiss 3T3 cells stimulated by bombesin, lysophosphatidic acid, and platelet-derived growth factor.

The experiments presented here were designed to examine the contribution of p125 focal adhesion kinase (p125FAK) tyrosine phosphorylation to the activation of the mitogen-activated protein kinase cascade induced by bombesin, lysophosphatidic acid (LPA), and platelet-derived growth factor (PDGF) in Swiss 3T3 cells. We found that tyrosine phosphorylation of p125FAK in response to these growth factors is completely abolished in cells treated with cytochalasin D or in cells that were suspended in serum-free medium for 30 min. In marked contrast, the activation of p42mapk by these factors was independent of the integrity of the actin cytoskeleton and of the interaction of the cells with the extracellular matrix. The protein kinase C inhibitor GF 109203X and down-regulation of protein kinase C by prolonged pretreatment of cells with phorbol esters blocked bombesin-stimulated activation of p42mapk, p90rsk, and MAPK kinase-1 but did not prevent bombesin-induced tyrosine phosphorylation of p125FAK. Furthermore, LPA-induced p42mapk activation involved a pertussis toxin-sensitive guanylate nucleotide-binding protein, whereas tyrosine phosphorylation of p125FAK in response to LPA was not prevented by pretreatment with pertussis toxin. Finally, PDGF induced maximum p42mapk activation at concentrations (30 ng/ml) that failed to induce tyrosine phosphorylation of p125FAK. Thus, our results demonstrate that p42mapk activation in response to bombesin, LPA, and PDGF can be dissociated from p125FAK tyrosine phosphorylation in Swiss 3T3 cells.

3T3 Cells↗
PubMed1995

Dissociation of cAMP-stimulated mitogenesis from activation of the mitogen-activated protein kinase cascade in Swiss 3T3 cells.

Elevation of intracellular cAMP by forskolin, 8-bromoadenosine 3':5'-cyclic monophosphate, and prostaglandin E1, in synergy with insulin, stimulated DNA synthesis in quiescent Swiss 3T3 cells to the same level achieved by platelet-derived growth factor (PDGF) or bombesin. Both forskolin and 8-bromoadenosine 3':5'-cyclic monophosphate stimulated a significant increase in cell number which, in the presence of insulin, reached the same levels achieved with PDGF. Treatment with either PDGF or bombesin caused a marked and persistent stimulation of p42MAPK and p44MAPK. In striking contrast, no activation was seen with mitogenic combinations of cAMP as shown by three different assays. Swiss 3T3 cells stably transfected with a constitutively activated Gs alpha subunit were 100-fold more sensitive to the mitogenic effects of forskolin but in this distinct cellular model forskolin did not activate p42MAPK. Swiss 3T3 cells stably transfected with interfering mutants of MEK-1 showed a 60% decrease in PDGF-stimulated p42 MAPK activation, but there was no inhibition of the mitogenic effect of forskolin in these cells. Furthermore, the upstream kinases MEK-1/MEK-2 and p74raf-1 were not activated by mitogenic combinations of cAMP while PDGF caused marked stimulation of their activity. Treatment of 3T3 cells with forskolin attenuated PDGF-stimulated p74raf-1 and p42MAPK activation but enhanced the mitogenic effects of this agent. Mitogenic combinations of cAMP strongly stimulated the phosphorylation and activation of p70s6k an effect that was inhibited by rapamycin. This agent markedly inhibited cAMP-stimulated DNA synthesis suggesting a critical role for p70s6k in cAMP mitogenic signaling. These results demonstrate that cAMP-induced mitogenesis can be dissociated from activation of the mitogen-activated protein kinase cascade and that this is not an obligatory point of convergence in mitogenic signaling in Swiss 3T3 cells.

3T3 Cells↗
PubMed1995

Technical note: inspiration or expiration? Reducing motion artefact in digital subtraction arch angiography of the extracranial carotid arteries.

100 consecutive patients underwent digital subtraction aortic arch angiography for suspected carotid bifurcation disease. Patients were prospectively randomized to suspend respiration in inspiration or expiration during image acquisition. Images were assessed qualitatively as "excellent", "adequate" or "poor". 92 studies were available for review. Adequate and poor groups were combined into a non-excellent group due to the small numbers that were scored poor. There were 27 (53%) excellent, 24 (47%) non-excellent in the expiration group and 12 (29%) excellent, 29 (71%) non-excellent in the inspiration group. Analysis of angiogram scores gave chi 2(1) = 4.3; p = 0.038, indicating that the higher angiogram scores of the expiration group were statistically significant. Pixel shifting was used in 61 cases (66.3%). The proportion was 34.4% lower in the expiration group (95% CI; 17-52%) with chi 2(1) = 10.5; p = 0.0012 indicating that the reduction in the use of pixel shifting for the expiration group was statistically significant. The superiority of arch angiograms obtained with the patient suspending respiration in expiration rather than inspiration has been demonstrated. This was achieved with significantly reduced use of pixel shifting, encouraging greater confidence in the validity of the angiographic findings. The technique is free of cost or time implications.

Adult↗
PubMed1995

The human CCKB/gastrin receptor transfected into rat1 fibroblasts mediates activation of MAP kinase, p74raf-1 kinase, and mitogenesis.

The human cholecystokinin (CCK)B/gastrin receptor was stably transfected into Rat1 fibroblasts to examine the signaling pathways mediated by this seven-transmembrane, G protein-linked receptor. We report here that binding of CCK-8 or gastrin to the CCKB/gastrin receptor induced phosphoinositide breakdown and led to a rapid, transient, and concentration-dependent increase in intracellular Ca2+, which was completely blocked by a specific CCKB receptor antagonist. The peptides also stimulated tyrosine phosphorylation of focal adhesion kinase (p125FAK) and paxillin. Both CCK-8 and gastrin induced a dose- and time-dependent activation of MAP kinase and p74raf-1 kinase in the transfected Rat1 cells. These effects could be dissociated from protein kinase C activation and were not dependent on a functional Gi protein. Finally, both CCK-8 and gastrin induced DNA synthesis in Rat1 cells transfected with the human CCKB/gastrin receptor through a pertussis toxin-insensitive pathway. These results indicate that the neuropeptides gastrin and CCK can activate multiple signal transduction pathways and act as sole mitogens by binding to the CCKB/gastrin receptor transfected into Rat1 fibroblasts.

Animals↗
PubMed1992

Evidence for neuromedin-B synthesis in the rat anterior pituitary gland.

Neuromedin-B shows a widespread distribution throughout mammalian neural and peripheral tissues and may be involved in the modulation of a variety of physiological processes. The highest concentrations of neuromedin-B are found in the anterior pituitary gland, suggesting that it may be of physiological importance within this tissue. To examine this hypothesis, we have used Northern blotting to demonstrate that neuromedin-B is present in the anterior pituitary as a result of local synthesis and have examined the effects of endocrine manipulations on its mRNA and immunoreactive peptide content. In thyroidectomized male rats, neuromedin-B content was decreased (104.1 +/- 5.8 vs. control, 390.9 +/- 23.3 fmol/gland; P less than 0.01) as was its mRNA (7 +/- 1.3% vs. 100%; P less than 0.014), while treatment of intact animals with T4 produced no effect on either peptide content or mRNA. Adrenalectomized male rats showed a significant increase in both neuromedin-B content (313.8 +/- 11.8 vs. control, 233.7 +/- 16.9 fmol/gland; P less than 0.05) and mRNA (377 +/- 27% vs. 100%; P less than 0.014), while dexamethasone treatment increased peptide content (347.8 +/- 32 vs. control, 233.7 +/- 16.9 fmol/gland; P less than 0.01) without any effect on mRNA levels. In female rats, ovariectomy decreased neuromedin-B content (132.4 +/- 13.3 vs. control, 335.0 +/- 37.2 fmol/gland; P less than 0.01) and mRNA levels (6 +/- 2% vs. 100%; P less than 0.014) while estrogen treatment of both ovariectomized and intact rats produced large increases in neuromedin-B (887.8 +/- 114.4 and 1328 +/- 175 fmol/gland, respectively, vs. control, 335.0 +/- 37.2 fmol/gland; both P less than 0.01) and its mRNA (246 +/- 18% and 378 +/- 31%, respectively, vs. 100%; both P less than 0.014). In castrated male rats, no significant alteration in peptide content was observed, and treatment of both castrated and intact male rats with testosterone was similarly without effect. These results demonstrate that 1) neuromedin-B is locally synthesized within the rat anterior pituitary gland; and 2) the local production of neuromedin-B is influenced by endocrine status, which is consistent with an autocrine/paracrine role for this peptide in this tissue.

Animals↗