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B L Fanburg

Publications and source records attributed to B L Fanburg.

At least 37 records · Page 2Linked to original sources
PubMed1998

Activation of the JAK-STAT pathway by reactive oxygen species.

Reactive oxygen species (ROS) play an important role in the pathogenesis of many human diseases, including the acute respiratory distress syndrome, Parkinson's disease, pulmonary fibrosis, and Alzheimer's disease. In mammalian cells, several genes known to be induced during the immediate early response to growth factors, including the protooncogenes c-fos and c-myc, have also been shown to be induced by ROS. We show that members of the STAT family of transcription factors, including STAT1 and STAT3, are activated in fibroblasts and A-431 carcinoma cells in response to H2O2. This activation occurs within 5 min, can be inhibited by antioxidants, and does not require protein synthesis. STAT activation in these cell lines is oxidant specific and does not occur in response to superoxide- or nitric oxide-generating stimuli. Buthionine sulfoximine, which depletes intracellular glutathione, also activates the STAT pathway. Moreover, H2O2 stimulates the activity of the known STAT kinases JAK2 and TYK2. Activation of STATs by platelet-derived growth factor (PDGF) is significantly inhibited by N-acetyl-L-cysteine and diphenylene iodonium, indicating that ROS production contributes to STAT activation in response to PDGF. These findings indicate that the JAK-STAT pathway responds to intracellular ROS and that PDGF uses ROS as a second messenger to regulate STAT activation.

3T3 Cells↗
PubMed1997

Association of Tyr phosphorylation of GTPase-activating protein with mitogenic action of serotonin.

We have previously shown that serotonin (5-HT) induces both hyperplasia and hypertrophy of pulmonary artery smooth muscle cells (SMC) but not of endothelial cells (EC) through its high-affinity uptake. The present studies demonstrate rapid enhancement by 5-HT of Tyr phosphorylation of proteins, including p120, which also occurs in SMC but not in EC. The p120 protein was identified as GTPase-activating protein (GAP) by immunoprecipitation. Its phosphorylation occurred within minutes and preceded other events associated with 5-HT-induced mitogenesis. Tyr kinase (TK) and 5-HT uptake inhibitors and 8-bromoadenosine 3',5'-cyclic monophosphate blocked both the 5-HT-induced DNA synthesis and Tyr phosphorylation of GAP. Vanadate elevated DNA synthesis and Tyr phosphorylation of GAP of both control and 5-HT-treated cells. 5-HT failed to alter Tyr phosphorylation of GAP in cellular homogenates, as opposed to intact cells. In the presence of 3-isobutyl-1-methylxanthine, 5-HT inhibited cellular growth, presumably through its action on 5-HT1A or 5-HT4 receptors and elevation of adenosine 3',5'-cyclic monophosphate, but this was not associated with an alteration of Tyr phosphorylation of GAP. Similarly, a 5-HT1 or 5-HT2 receptor agonist failed to stimulate Tyr phosphorylation or DNA synthesis of SMC. Stimulation of cellular proliferation and enlargement produced by 1 microM 5-HT were totally abolished by TK inhibitors that did not affect 5-HT uptake. These data indicate that Tyr phosphorylation of GAP may act as an intermediate signal in 5-HT-induced mitogenesis of SMC which requires cellular internalization of 5-HT rather than its action on a membrane receptor.

1-Methyl-3-isobutylxanthine↗
PubMed1997

A new role for an old molecule: serotonin as a mitogen.

Serotonin (5-hydroxytryptamine) has been known for the last half century to influence vasoactivity and to participate in neurotransmission. More recently, this compound has been recognized to cause proliferation of a variety of cells in culture, including those of vascular smooth muscle. Furthermore, the proliferative effect is synergistic with that of more conventional growth-producing polypeptides. A hypertrophic, as well as a proliferative response, has been shown to occur in some smooth muscle cells. Whether the mitogenic effect is initiated through a cell surface receptor or a serotonin transporter, or both depending on the cell type, is currently unresolved. Most evidence indicates that cellular cyclic nucleotides play an important role in the intracellular signaling process for growth regulation by serotonin, and newer studies point to protein phosphorylation pathways as being important in the mitogenic response. It has been proposed that, through these signaling pathways, serotonin plays an important role in remodeling of both the pulmonary and systemic circulations.

Animals↗
PubMed1997

Inhibitory effect of heparin on serotonin-induced hyperplasia and hypertrophy of smooth muscle cells.

Serotonin (5-HT) produces both hyperplastic and hypertrophic effects on smooth muscle cell (SMC) in culture. Heparin is known to inhibit serum-induced hyperplasia of SMC but has not been previously tested on the stimulatory effect of 5-HT on SMC. Our present data show that at 24 h heparin inhibited by 50% the stimulation of 3H-thymidine incorporation into bovine pulmonary artery SMC and at 7 days totally reversed both cellular proliferation and enlargement of SMC produced by 1 microM 5-HT. Heparin failed to alter 5-HT uptake by SMC, but inhibited the stimulation of tyrosine phosphorylation of GTPase-activating protein, a proposed intermediate in the 5-HT stimulatory process. Thus heparin inhibits both hyperplastic and hypertrophic effects of 5-HT on SMC, perhaps through the inhibition of a phosphorylated intermediate protein.

Animals↗
PubMed1997

Transforming growth factor B1 decreases uptake of glutathione precursor amino acids in bovine pulmonary artery endothelial cells.

We have previously observed that transforming growth factor beta 1 (TGF beta 1) produces a pro-oxidant effect and decreases cellular glutathione (GSH) levels of cultured bovine pulmonary artery endothelial cells (BPAEC) (White A. C., S. K. Das, and B. L. Fanburg. Am. J. Respir. Cell Mol. Biol. 6:364-368, 1992). In the present studies we demonstrate that 2 ng/ml TGF beta 1 reduces the uptake of two GSH precursor amino acids (cystine and glutamate) by 50% (cystine; control 359.35 +/- 100, TGF beta 1 187.7 +/- 26 pmol/10 min/10(6) cells, p < 0.05; glutamate; control 215.15 +/- 18, TGF beta 1 110.2 +/- 16 pmol/10 min/10(6) cells, p < 0.001). The inhibitory effect of TGF beta 1 on the uptake of GSH precursor amino acids persisted in the presence of buthionine sulfoximine (inhibits gamma-glutamyl cysteine synthetase, the rate limiting step in GSH synthesis) or acivicin (inhibits gamma-glutamyl transpeptidase). The uptake of leucine, an amino acid that does not serve as a precursor for GSH, was unaffected by TGF beta 1. In additional experiments TGF beta 1 decreased the levels of cellular and medium GSH-indicating that TGF beta 1 did not increase efflux of GSH from BPAEC. We propose from these observations that TGF beta 1 decreases cellular glutathione, at least in part, through down regulation of precursor amino acid transport and, thereby, its rate of synthesis.

Animals↗
PubMed1996

Nitroprusside inhibits serotonin-induced mitogenesis and tyrosine phosphorylation of smooth muscle cells.

Serotonin (5-hydroxytryptamine, 5-HT) produces hyperplasia and hypertrophy of bovine pulmonary artery smooth muscle cells (SMC) in culture. The growth responses are associated with early elevations of c-myc and actin gene expressions and are blocked by agents that elevate cellular adenosine 3',5'-cyclic monophosphate or inhibit 5-HT transport or tyrosine phosphorylation. A rapid enhancement of tyrosine phosphorylation of a 120-kDa protein (p120) is associated with the 5-HT-induced mitogenesis. In the present studies, sodium nitroprusside (SNP, 10-100 micromol/l), a NO-generating agent, dose dependently inhibited 5-HT-induced thymidine incorporation by SMC. Inhibition of the 5-HT stimulatory effect was also observed with isosorbide dinitrate and nitroglutathione, which are also NO donors. Incubation of cells with 8-bromoguanosine 3',5'-cyclic monophosphate (1 micromol/l) mimicked the antimitogenic action of SNP. The antiproliferative effect of SNP was inhibited by hemoglobin (50 micromol/l) and potentiated by superoxide dismutase (200 U/ml), supporting the role of NO in the process. Enhancement of tyrosine phosphorylation of p120 by 5-HT was prevented by preincubation with SNP or exogenously added guanosine 3',5'-cyclic monophosphate. The data indicate that 5-HT acts as a mitogen for SMC through a signal transduction pathway involving tyrosine phosphorylation. SNP likely prevents the 5-HT-induced mitogenesis of SMC through elevation of intracellular guanosine 3',5'-cyclic monophosphate and inhibition of tyrosine phosphorylation of p120.

Actins↗
PubMed1995

Activation of an H2O2-generating NADH oxidase in human lung fibroblasts by transforming growth factor beta 1.

The cellular source(s) and mechanisms of generation of reactive oxygen species (ROS) in nonphagocytic cells stimulated by cytokines are unclear. In this study, we demonstrate that transforming growth factor beta 1 (TGF-beta 1, 1 ng/ml) induces the release of H2O2 from human lung fibroblasts within 8 h following exposure to this cytokine. Elevation in H2O2 release peaked at 16 h (approximately 22 pmol/min/10(6) cells) and gradually declined to undetectable levels at 48 h after TGF-beta 1 treatment. NADH consumption by these cells was stimulated by TGF-beta 1 while that of NADPH remained unchanged. NADPH oxidase activity as measured by diphenyliodonium (DPI)-inhibitable NADH consumption in TGF-beta 1-treated cells followed a time course similar to that of H2O2 release. DPI, an inhibitor of the NADPH oxidase complex of neutrophils and other flavoproteins, also inhibited the TGF-beta 1-induced H2O2 production. Inhibitors of other enzymatic systems involving flavoproteins that may be responsible for the production of H2O2 in these cells, including xanthine oxidase, nitric oxide synthase, and both mitochondrial and microsomal electron transport systems, failed to inhibit TGF-beta 1-induced NADH oxidation and H2O2 production. The delay (> 4 h) following TGF-beta 1 exposure along with the inhibition of this process by cycloheximide and actinomycin D suggest the requirement of new protein synthesis for induction of NADH oxidase activity in TGF-beta 1-stimulated fibroblasts.

Animals↗
PubMed1995

Nitric oxide increases cellular glutathione levels in rat lung fibroblasts.

Nitric oxide (NO) has been demonstrated to play a protective role in cell injury. In this study, we have explored the effect of NO and two NO donors (sodium nitroprusside [SNP] and isosorbide dinitrate [ISDN]) on cellular glutathione (GSH) levels in a rat lung fibroblast cell line (RFL6 cells). SNP and ISDN significantly increased cellular GSH in RFL6 cells (5 x 10(-4) M SNP: 21.9 +/- 3.6 nmol/10(6) cells and 5 x 10(-3) M ISDN: 27.6 +/- 1.7 nmol/10(6) cells versus control: 13.2 +/- 0.4 nmol/10(6) cells; P < 0.05). The stimulatory effect of SNP and ISDN on GSH was first seen at 6 h and peaked at 12 to 24 h. A similar increase in GSH was observed in RFL6 cells exposed to 400 ppm NO for 7.5 h (NO: 20.5 +/- 3.4 nmol/10(6) cells versus control: 11.9 +/- 2.4; P < 0.05). SNP and ISDN also increased cellular GSH in bovine pulmonary artery smooth muscle cells (BPSMC) and bovine pulmonary artery endothelial cells (BPAEC). Buthionine sulfoximine (BSO) (0.01 mM), an inhibitor of the GSH synthetic enzyme gamma-glutamyl cysteine synthetase, blocked the increase in GSH in RFL6 cells seen with both SNP and ISDN. In BPAEC, exposure to NO donors for 24 h stimulated glutamate uptake (SNP: 441 +/- 19 pmol/10 min/10(6) cells and ISDN: 677 +/- 48 pmol/10 min/10(6) min/10(6) cells versus control: 222 +/- 9 pmol/10 min/10(6); P < 0.05). This effect paralleled the increase in GSH. In RFL6 cells, only SNP increased glutamate uptake after 24 h of incubation. In summary, NO and NO donors increase cellular GSH in RFL6 cells, BPAEC, and BPSMC. The mechanism of this effect is unclear but may involve upregulation of the normal GSH synthetic pathways. This observation may explain in part the protective effect of NO seen in some cell culture systems and may contribute to a protective effect against oxidant injury in vivo.

Animals↗
PubMed1994

Angiotensin converting enzyme (kininase II) mRNA production and enzymatic activity in human peripheral blood monocytes are induced by GM-CSF but not by other cytokines.

Peripheral blood monocytes (PBM) do not possess angiotensin converting enzyme (ACE) activity in the inactive state. However, measurable PBM ACE activity is found in patients with certain inflammatory diseases. We have examined the effect of cytokines likely to be present during granulomatous inflammation on the regulation of ACE mRNA in PBM. The presence of ACE mRNA in human PBM cultured in vitro with various cytokines for up to 6 days was analyzed using polymerase chain reaction. PBM not exposed to cytokines did not express ACE mRNA, while incubation of PBM with recombinant human GM-CSF resulted in high levels of ACE mRNA expression after 72 h of cell culture, which persisted through day six. Increased ACE mRNA expression occurred concomitantly with phenotypic changes in cell size and shape consistent with cell activation. A 5-fold increase in ACE enzymatic activity also occurred. Incubation of PBM with all other cytokines tested failed to induce ACE mRNA expression. Alveolar macrophages expressed ACE mRNA immediately following their isolation, but mRNA expression decreased markedly during a 24-h period of incubation and was only partially reversed with exogenous GM-CSF. We conclude that GM-CSF enhances ACE mRNA levels in human PBM, but not in alveolar macrophages.

Base Sequence↗
PubMed1994

Relative quantification of angiotensin-converting enzyme mRNA in human smooth muscle cells, monocytes, and lymphocytes by the polymerase chain reaction.

A method was developed for relative quantification of angiotensin-converting enzyme (ACE) mRNA in as few as 100 cells. After reverse transcription of total RNA to cDNA, multiplexed polymerase chain reaction with two sets of primers amplified ACE cDNA and that of an internal standard glyceraldehyde phosphate dehydrogenase (GAPDH) simultaneously. By adjusting primer pair concentrations, both ACE and GAPDH were amplified with constant efficiencies. Macrophage-like U937 histiocytic lymphoma cells expressed ACE mRNA constitutively. Freshly isolated human monocytes did not express ACE mRNA initially, but after 4 days in culture had 8% of the amount found in U937 cells. After phorbol ester stimulation, monocytes transcribed ACE at levels comparable to U937 cells. Human smooth muscle cells had ninefold more ACE mRNA than 4-day monocytes, but 30% less than U937 cells. In contrast, a mixed population of lymphocytes was devoid of ACE mRNA.

Base Sequence↗
PubMed1994

A competitive deletion mutant quantitative PCR assay for angiotensin-converting enzyme mRNA in smooth muscle cells.

To quantify angiotensin-converting enzyme (ACE) mRNA, we have developed a reverse transcription (RT)-coupled competitive PCR (RT-PCR) assay with a deletion mutant internal standard. The RT-PCR detects ACE mRNA from both human and bovine sources. ACE mRNA was detected in total RNA from cultured human saphenous vein smooth muscle cells (HuSV-SMCs) and from bovine pulmonary artery (BPA) SMCs. BPA-SMC expressed ninefold less ACE mRNA than BPA endothelial cells, and threefold less than HuSV-SMCs. Apparent amounts of ACE mRNA were 118,350 +/- 2,300 copies in HuSV-SMCs and 42,200 +/- 11,300 copies in BPA-SMCs per microgram of total cell RNA. The accuracy of the absolute values is subject to the limitations of the assumptions used to calculate them. These data support the hypothesis that components of the renin-angiotensin system are transcribed by SMCs.

Animals↗
PubMed1994

Serotonin produces both hyperplasia and hypertrophy of bovine pulmonary artery smooth muscle cells in culture.

Serotonin [5-hydroxytryptamine (5-HT)] has a dual effect on bovine pulmonary artery smooth muscle cells (SMC) in culture (S.-L. Lee, W. W. Wang, B. J. Moore, and B. L. Fanburg. Circ. Res. 68: 1362-1368, 1991.). Cellular internalization of 5-HT stimulates DNA synthesis and cellular proliferation, whereas the action of 5-HT on a cell surface receptor elevates adenosine 3',5'-cyclic monophosphate and inhibits proliferation. The present study shows that 5-HT causes proliferation of both pulmonary artery and aortic SMC but not of endothelial cells or fibroblasts. Furthermore, c-myc and alpha- and beta-actin gene expressions of pulmonary artery SMC were elevated after 2-h incubation with 5-HT, before stimulation of [3H]thymidine incorporation. Actinomycin D (0.05 micrograms/ml) but not cycloheximide (1 microgram/ml) inhibited the gene expression produced by 5-HT. Growth-arrested SMC progressed from a G0 quiescent state through a normal cell cycle when subjected to 5-HT, 5-HT plus 25 ng/ml insulin-like growth factor, or 5-HT plus 0.5 ng/ml fibroblast growth factor. Cell number increased by 20-40% at 40 h and 50-140% at 7 days. Protein content of cells treated with 5-HT was elevated by 20-40% at 7 days, whereas the rate of protein synthesis, measured by [35S]methionine incorporation, increased by 50-70% at 24 h. In the presence of 1 microM 5-HT, cells enlarged by 70 and 100-200% at 40 h and 7 days, respectively.(ABSTRACT TRUNCATED AT 250 WORDS)

Actins↗
PubMed1994

Regulation of serotonin-induced DNA synthesis of bovine pulmonary artery smooth muscle cells.

We have previously reported that serotonin [5-hydroxytryptamine (5-HT)] stimulates DNA synthesis of bovine pulmonary artery smooth muscle cells (SMC) by its high-affinity uptake. Uptake inhibitors, but not selective 5-HT receptor antagonists, prevented the stimulatory effect (S.-L. Lee and B. L. Fanburg. J. Cell. Physiol. 150:396-405, 1992). We have now further evaluated the mechanism by which 5-HT enhances SMC DNA synthesis. Although some serotonergic agonists mimicked this stimulation, selective 5-HT receptor agonists produced no or only minor and variable stimulatory effects. The action of 5-HT was not inhibited by inhibitors of phospholipases C and A2, the protein kinase C (PKC) inhibitors dihydrosphingosine and 1-(-isoquinolinylsulfonyl)-2 methylpiperazine (H-7), or down-regulation of PKC with phorbol 12,13-dibutyrate. Staurosporine, a reputed PKC and tyrosine kinase (TK) inhibitor, and genistein, a selective TK inhibitor, reversed the stimulatory effect of 5-HT in a dose-dependent manner. Before stimulation of thymidine incorporation into cellular DNA, 5-HT elevated c-myc and actin mRNAs. Imipramine, fluoxetine, staurosporine, and cholera toxin inhibited the stimulations of both DNA synthesis and c-myc and actin mRNA expressions by 5-HT. Thus the data support a concept that 5-HT-induced thymidine incorporation by SMC involves membrane transport of 5-HT that initiates tyrosine phosphorylation.

Actins↗
PubMed1994

Regulation of endothelial cell xanthine dehydrogenase xanthine oxidase gene expression by oxygen tension.

Recent studies have demonstrated that xanthine dehydrogenase/xanthine oxidase (XD/XO) activities of bovine endothelial cells (EC) are inversely regulated by O2 tensions to which the cells are exposed. We have confirmed these reports and extended the observation to a variety of cells from other sources. All EC that had detectable XD/XO activity demonstrated the greatest activity at the lowest O2 level. Bovine pulmonary artery smooth muscle cells showed XD/XO activity only under hypoxic conditions. The ratio of XO to XO+XD did not change significantly under various O2 concentrations for all cell types tested. Treatment of bovine pulmonary artery and rat epididymal fat pad EC with actinomycin D (1 microgram/ml), an inhibitor of transcription, suppressed XO and XO+XD activities in cells exposed both to 20 and 3% O2. High-dose cycloheximide (5 micrograms/ml), an inhibitor of translation, also reduced XO and XO+XD activities in these cells, whereas low-dose cycloheximide (0.5 microgram/ml) enhanced the stimulatory effect of hypoxia on XO+XD activity. We developed a digoxigenin-labeled probe that recognizes and hybridizes to rat XD cDNA and used it to examine the effect of O2 concentration on XD/XO mRNA expression of rat epididymal fat pad EC. XD/XO mRNA concentration was increased in cells exposed to hypoxia and decreased in cells exposed to hyperoxia compared with normoxic cells. The increase in mRNA concentration resulting from exposure to hypoxia was enhanced by cycloheximide. There was no change in XD/XO mRNA stability in cells exposed to hypoxia compared with normoxia. We conclude that the regulation of XD/XO by oxygen tension most likely occurs at the transcriptional level.

Adipose Tissue↗
PubMed1994

Respiratory dysfunction in myasthenia gravis.

Myasthenia gravis can affect the respiratory system, causing respiratory muscle weakness, an abnormal breathing pattern, and blunted ventilatory responses. Specific treatment can reverse most of these effects and prevent the development of respiratory failure.

Diagnosis, Differential↗
PubMed1993

Cu,Zn superoxide dismutase in vascular cells: changes during cell cycling and exposure to hyperoxia.

Conditions that generate reactive oxygen species elevate Cu,Zn superoxidase dismutase (SOD) in endothelial cells (EC) concomitant with decreased cellular proliferation. The current studies were undertaken with both vascular EC and smooth muscle cells (SMC) to compare the influences of cellular proliferation with those of hyperoxia on induction of Cu,Zn SOD. To assess cell cycling alone, EC and SMC were growth arrested, then released from arrest. Cell cycling was monitored by [3H]thymidine incorporation, counts, and flow cytometry. SOD catalytic activity was measured spectrophotometrically and SOD protein by enzyme-linked immunosorbent assay. A digoxigenin-labeled probe was used to quantify SOD mRNA by Northern analysis. EC reached the S phase of the cell cycle in 18 h and completed one cycle in 24-30 h, whereas SMC took 24-30 h to reach the S phase and 48 h to complete one cycle. Cu,Zn SOD mRNA for both EC and SMC was very low during the Go/G1 phase, peaked during the S phase, and then reverted to lower values as cells progressed through their cycles. Cu,Zn SOD activity and immunoprotein content showed corresponding changes to those of mRNA. Exposure to hyperoxia (95% O2) delayed the entry of released cells into the S phase of the cell cycle and blocked the cells in the S or G2 phase, but induced Cu,Zn SOD mRNA before the S phase and caused persistence of elevation of Cu,Zn SOD mRNA as cells progressed through their cycles. Exposure to hyperoxia also induced Cu,Zn SOD mRNA in growth-arrested cells within 24-48 h. Thus our studies support roles for both cells cycle dependency and reactive oxygen species in the induction of Cu,Zn SOD.

Animals↗
PubMed1993

Enhanced rate of H2O2 release from bovine pulmonary artery endothelial cells induced by TGF-beta 1.

We have previously reported that transforming growth factor-beta 1 (TGF-beta 1) produces a "prooxidant" effect on cultured bovine pulmonary artery endothelial cells (BPAEC) [S. K. Das and B. L. Fanburg. Am. J. Physiol. 261 (Lung Cell. Mol. Physiol. 5): L249-L254, 1991]. This effect was found to be associated with a lowering of total cellular GSH (A.C. White, S. K. Das, and B.L. Fanburg. Am. J. Respir. Cell Mol. Biol. 6: 364-368, 1992). In this study, we demonstrate a twofold increase in the rate of extracellular H2O2 release from BPAEC after a 72-h exposure to TGF-beta 1 (2 ng/ml, added at times 0 and 48 h). Increasing and decreasing the levels of cellular GSH with diethylmaleate (DEM, 0.05 mM) and buthionine sulfoximine (BSO, 0.01 mM), respectively, did not affect the rate of TGF-beta 1-induced increase in H2O2 release when compared with the individual effects of these reagents on control cells. The addition of BSO (0.01 mM) to control cells failed to demonstrate an increase in the rate of H2O2 release, despite a more profound decrease in cellular GSH by these cells than detected in cells treated with TGF-beta 1 alone. Moreover, a single dose of TGF-beta 1 (2 ng/ml) induced a 63-85% increase in the rate of H2O2 release within 16 h of exposure, well before the previously demonstrated lowering of cellular GSH. These results indicate that the increase in H2O2 production by TGF-beta 1-stimulated BPAEC is associated with, but does not appear to be the result of, a lowering of cellular GSH. This study further suggests that the TGF-beta 1-induced H2O2 production occurs at a site inaccessible to detoxification by GSH.

Analysis of Variance↗