scholarly journals Alpha 2-adrenergic agonists stimulate DNA synthesis in Chinese hamster lung fibroblasts transfected with a human alpha 2-adrenergic receptor gene.

1990 ◽  
Vol 1 (6) ◽  
pp. 445-451 ◽  
Author(s):  
K Seuwen ◽  
I Magnaldo ◽  
B K Kobilka ◽  
M G Caron ◽  
J W Regan ◽  
...  
Keyword(s):  
Cell Proliferation ◽  
Growth Factor ◽  
Dna Synthesis ◽  
Adrenergic Receptors ◽  
Adrenergic Receptor ◽  
Inositol Phosphates ◽  
Receptor Gene ◽  
Chinese Hamster ◽  
Lung Fibroblasts ◽  
Ccl39 Cells

To test the hypothesis that agents activating receptors negatively coupled to adenylyl cyclase (AC) can stimulate cell proliferation, we have expressed a human alpha 2-adrenergic receptor (alpha 2-C10) in CCL39 cells and studied the effects of alpha 2-agonists on reinitiation of DNA synthesis in quiescent cells. We report that the alpha 2-agonists epinephrine and clonidine stimulate [3H]-thymidine incorporation in synergy with fibroblast growth factor and that the alpha 2-antagonist yohimbine efficiently inhibits this response. Epinephrine- and clonidine-stimulated DNA synthesis is completely blocked by pertussis toxin and correlates well with the inhibition of prostaglandin E1-stimulated AC. Thus, their action closely resembles the action of serotonin in the same cell system, which is mediated through 5-HT1b receptors. In fact, serotonin- and epinephrine-stimulated DNA synthesis reinitiation is not additive, suggesting that both agents act through a common pathway. Interestingly, alpha 2-agonists also induced a moderate release of inositol phosphates, indicating that alpha 2-adrenergic receptors can interact both with the AC and phospholipase C messenger system. Activation of phosphoinositide (PI) turnover by epinephrine leads to a significant stimulation of Na+/H+ exchange but is insufficient to trigger a mitogenic response in CCL39 cells, as will be discussed. We found no evidence for epinephrine-induced activation of Na+/H+ exchange by a mechanism independent of PI breakdown.Our data show that alpha 2-adrenergic receptors can play a role in the regulation of cell proliferation in an appropriate context; also, the data support the hypothesis that receptors negatively coupled to AC must be taken into account as mediators of growth factor action in fibroblasts, in particular when activated in parallel with receptor tyrosine kinases.

Phospholipase C-β1 mediates α1-adrenergic receptor-stimulated activation of the sodium–hydrogen exchanger in Chinese hamster lung fibroblasts (CCL39)

2005 ◽  
Vol 83 (2) ◽  
pp. 123-132 ◽  
Author(s):  
J J Provost ◽  
S M Olmschenk ◽  
A L Metcalf ◽  
N Korpi ◽  
H Thronson ◽  
...  
Keyword(s):  
Phospholipase C ◽  
Cell Lines ◽  
Adrenergic Receptor ◽  
Chinese Hamster ◽  
Lung Fibroblasts ◽  
Specific Chemical ◽  
Sodium Hydrogen ◽  
Sodium Hydrogen Exchanger ◽  
Nhe1 Activity ◽  
Ccl39 Cells

The activation of the Na+–H+ exchanger 1 (NHE1) and extracellular-signal regulated kinase (ERK) phospho rylation in Chinese hamster lung fibroblasts (CCL39) was characterized in response to the specific α1-adrenergic agonist, phenylephrine (PE). Addition of 100 µmol PE/L increased the steady-state intracellular pH (pHi) by 0.16 ± 0.03 pH units, as well as increasing the phosphorylation of ERK. The response of NHE1 to PE in CCL39 cells was determined by the use of specific antagonists. Use of 2 specific chemical inhibitors of phosphoinositide-specific phospholipase C (PLC) reduced the ability of PE to activate either the exchanger or ERK. Studies were conducted in PLCβ-deficient cell lines derived from parental CCL39 cells. NHE1 activity in both mutant cell lines was increased in response to phorbal esters or lysophosphatidic acid, whereas the addition of PE only caused a minimal change in either pHi or ERK phosphorylation. These results, combined with reconstitution experiments with exogenously expressed PLCβ1, PLCβ2, or PLCβ3, revealed that stimulation of NHE1 activity by PE in CCL39 cells is a PLCβ1-coupled event. Furthermore, the data indicate that α1-adrenergic signaling of PLCβ is upstream of ERK activation. These data demonstrate that PLCβ1 is primarily involved in the activation of NHE1 in CCL39 fibroblasts.Key words: CCL39, sodium hydrogen exchanger, ERK, α1-adrenergic receptor, phospholipase Cβ.

Effects of epidermal growth factor on diamine oxidase expression and cell growth in Caco-2 cells

1991 ◽  
Vol 261 (4) ◽  
pp. G669-G676 ◽  
Author(s):  
B. Daniele ◽  
A. Quaroni
Keyword(s):  
Cell Proliferation ◽  
Growth Factor ◽  
Epidermal Growth Factor ◽  
Dna Synthesis ◽  
Intestinal Mucosa ◽  
Diamine Oxidase ◽  
Small Intestinal Mucosa ◽  
Cell Proliferation And Differentiation ◽  
Proliferation And Differentiation ◽  
Epidermal Growth

To investigate the role of diamine oxidase (DAO) in the intestinal mucosa, we compared its expression with cell proliferation and differentiation in the human colon carcinoma cell line Caco-2. DAO synthesis was evaluated in subconfluent and confluent cultures and in the presence of epidermal growth factor (EGF), a polypeptide hormone known to have specific trophic effects on the small intestinal mucosa. EGF stimulated DNA synthesis, significantly increased cellular DAO activity and the amount of enzyme secreted into the culture medium, but decreased expression of dipeptidyl peptidase IV, a marker of cell differentiation in confluent Caco-2 cells. Immunoprecipitation of DAO from cells labeled metabolically with [35S]methionine failed to demonstrate an increased enzyme synthesis in EGF-treated cells, suggesting that this hormone acted primarily at a posttranslational level by reducing DAO degradation before intracellular storage or secretion. A possible relationship between changes in cellular DAO activity and cell proliferation was also investigated by using aminoguanidine, a specific and potent DAO inhibitor. Although DAO activity was markedly suppressed, aminoguanidine had no significant effects on the rate of DNA synthesis. These results demonstrated that in Caco-2 cells EGF stimulated DNA synthesis and DAO expression; however, cell proliferation and differentiation were not correlated with the levels of cellular DAO, suggesting that this enzyme does not play a major role in the regulation of intestinal epithelial cell turnover.

scholarly journals Effects of pertussis toxin on growth factor-stimulated inositol phosphate formation and DNA synthesis in Swiss 3T3 cells

1988 ◽  
Vol 249 (3) ◽  
pp. 917-920 ◽  
Author(s):  
C W Taylor ◽  
D M Blakeley ◽  
A N Corps ◽  
M J Berridge ◽  
K D Brown
Keyword(s):  
Growth Factor ◽  
Dna Synthesis ◽  
Pertussis Toxin ◽  
Inositol Phosphates ◽  
Inositol Phosphate ◽  
Phosphoinositide Hydrolysis ◽  
Polypeptide Growth Factors ◽  
Swiss 3T3 ◽  
Swiss 3T3 Cell ◽  
Stimulation Of

We have compared the effects of pretreatment of Swiss 3T3 cell with pertussis toxin on the stimulation of DNA synthesis and phosphoinositide hydrolysis in response to a wide variety of mitogens. The toxin substantially inhibited the stimulation of DNA synthesis in response to a phorbol ester or various peptide and polypeptide growth factors irrespective of their ability to activate phosphoinositidase C. Production of inositol phosphates in response to platelet-derived growth factor, fibroblast growth factor and prostaglandin F2 alpha were unaffected by the toxin while bombesin- and vasopressin-stimulated formation of inositol phosphates were inhibited by only 27 and 23% respectively. These results argue against a major role for a pertussis toxin-sensitive G protein in coupling any of these mitogen receptors to activation of a phosphoinositidase C. Furthermore, the results suggest that the widespread inhibitory effects of pertussis toxin on mitogen-stimulated DNA synthesis may be unrelated to the toxin's limited actions on phosphoinositide hydrolysis.

scholarly journals Internalization of the opioid growth factor, [Met5]-enkephalin, is dependent on clathrin-mediated endocytosis for downregulation of cell proliferation

2010 ◽  
Vol 299 (3) ◽  
pp. R774-R785 ◽  
Author(s):  
Fan Cheng ◽  
Patricia J. McLaughlin ◽  
William A. Banks ◽  
Ian S. Zagon
Keyword(s):  
Cell Proliferation ◽  
Growth Factor ◽  
Dna Synthesis ◽  
Wound Repair ◽  
Opioid Antagonist ◽  
Live Cells ◽  
Inhibitory Peptide ◽  
African Green Monkey Kidney ◽  
Initial Exposure ◽  
Opioid Growth Factor

The opioid growth factor (OGF; [Met5]-enkephalin), a constitutively expressed and tonically active inhibitory peptide, interacts with the OGF receptor (OGFr) to form an endogenous growth-regulating pathway in homeostasis. Amplification of OGF-OGFr interfacing in animal and clinical studies depresses development, neoplasia, angiogenesis, and immunity. Disruption of the OGF-OGFr axis accelerates cell proliferation and has been particularly important in wound repair. To investigate how OGF enters cells, OGF was labeled with 5,6-tetramethylrhodamine OGF (RhoOGF) to study its uptake in live cells. African green monkey kidney cells (COS-7) incubated with RhoOGF exhibited a temperature-dependent course of entry, being internalized at 37°C but not at 4°C. RhoOGF was detected in the cytoplasm 15 min after initial exposure, observed in both cytoplasm and nucleus within 30 min, and remained in the cells for as long as 5 h. A 100-fold excess of OGF or the opioid antagonist naltrexone, but not other opioid ligands (some selective for classic opioid receptors), markedly reduced entry of RhoOGF into cells. RhoOGF was functional because DNA synthesis in cells incubated with RhoOGF (10−5 to 10−8 M) was decreased 24–36%, and was comparable to cells treated with unlabeled OGF (reductions of 26–39%). OGF internalization was dependent on clathrin-mediated endocytosis, with addition of clathrin siRNA diminishing the uptake of RhoOGF and upregulating DNA synthesis. RhoOGF clathrin-mediated endocytosis was unrelated to endosomal or Golgi pathways. Taken together, these results suggest that OGF enters cells by active transport in a saturable manner that requires clathrin-mediated endocytosis.

Mediators of the mitogenic action of human V1vascular vasopressin receptors

2000 ◽  
Vol 279 (5) ◽  
pp. H2529-H2539 ◽  
Author(s):  
Marc Thibonnier ◽  
Doreen M. Conarty ◽  
Christine L. Plesnicher
Keyword(s):  
Cell Cycle ◽  
Cell Proliferation ◽  
Protein Kinase ◽  
Dna Synthesis ◽  
Cell Cycle Progression ◽  
Tritiated Thymidine ◽  
Chinese Hamster ◽  
Mitogen Activated Protein Kinase ◽  
Thymidine Uptake ◽  
Mitogenic Action

Arginine vasopressin (AVP) activation of V1 vascular receptors (V1Rs) stimulates cell growth and proliferation in different tissues via cellular signaling pathways that remain to be identified. To explore the intracellular mediators of the mitogenic action of V1R, Chinese hamster ovary (CHO) cells were stably transfected with the human V1R cDNA clone we isolated previously. We assessed AVP effects on kinase activation (immunoblotting with phosphospecific antibodies), DNA synthesis (tritiated thymidine uptake), cell cycle progression (flow cytometry analysis after nuclear labeling with propidium iodide), and cell proliferation (conversion of the colorimetric reagent MTS) in the presence or absence of various pathway inhibitors. AVP stimulation of V1Rs leads to the phosphorylation of several kinases, an increase in DNA synthesis, a progression through the S and G2–M phases of the cell cycle, and an increase in cell proliferation. The mediators of the mitogenic action of V1R activation included calcium mobilization, coupling to a Gq protein, and the simultaneous and parallel activation of several kinases, mainly calcium/calmodulin-dependent kinase II, phosphatidylinositol 3 kinase, protein kinase C, and p42/p44 mitogen-activated protein kinase.

Growth factor stimulated cell proliferation is accompanied by an elevated labile intracellular pool of zinc in 3T3 cells

2002 ◽  
Vol 80 (8) ◽  
pp. 790-795 ◽  
Author(s):  
Shirley C Paski ◽  
Zhaoming Xu
Keyword(s):  
Cell Proliferation ◽  
Growth Factors ◽  
Growth Factor ◽  
Dna Synthesis ◽  
Cell Number ◽  
3T3 Cells ◽  
Intracellular Pool ◽  
Igf I ◽  
Epidermal Growth ◽  
Number Counts

Growth factors such as platelet-derived growth factor (PDGF), epidermal growth factor (EGF), and insulin-like growth factor-I (IGF-I) are required for quiescent 3T3 cells to proliferate, but zinc deprivation impairs IGF-I-induced DNA synthesis. We recently showed that labile intracellular pool of zinc is involved in cell proliferation. Our objective was to determine whether the labile intracellular pool of zinc plays a role in growth factor (PDGF, EGF, and IGF-I) - stimulated proliferation of 3T3 cells. Quiescent 3T3 cells were cultured in DMEM with or without growth factors. Labile intracellular pool of zinc, DNA synthesis, and cell proliferation were assessed using fluorescence microscopy, 3H-thymidine incorporation, and total cell number counts, respectively. After 24 h, growth factors stimulated DNA synthesis (24%) but not cell proliferation. After 48 h, growth factors stimulated both DNA synthesis (37%) and cell proliferation (89%). In response to growth factor stimulation, the labile intracellular pool of zinc was also elevated after 24 or 48 h of treatment. In summary, growth factor (PDGF, EGF, and IGF-I) - stimulated increase in DNA synthesis and cell proliferation were accompanied by an elevated labile intracellular pool of zinc in 3T3 cells. Since elevation of the labile intracellular pool of zinc occurred along with increased DNA synthesis, but cell proliferation remained unchanged, the elevation of the labile intracellular pool of zinc likely occurred during the S phase to provide the zinc needed to support DNA synthesis and ultimately cell proliferation.Key words: PDGF, EGF, IGF-I, labile intracellular pool of zinc, cell proliferation, DNA synthesis, 3T3 cells.

Involvement of PDGF in pressure-induced mesangial cell proliferation through PKC and tyrosine kinase pathways

1999 ◽  
Vol 277 (1) ◽  
pp. F105-F112 ◽  
Author(s):  
Hiroaki Kato ◽  
Akihiko Osajima ◽  
Yasuhito Uezono ◽  
Masahiro Okazaki ◽  
Yuki Tsuda ◽  
...  
Keyword(s):  
Cell Proliferation ◽  
Protein Kinase C ◽  
Growth Factor ◽  
Protein Kinase ◽  
Tyrosine Kinase ◽  
Dna Synthesis ◽  
Tyrosine Kinases ◽  
Mechanical Stretch ◽  
Transmural Pressure ◽  
Kinase C

In glomerular hypertension, mesangial cells (MC) are subjected to at least two physical forces: mechanical stretch and high transmural pressure. Increased transmural pressure, as well as mechanical stretch, promotes MC proliferation, which may enhance glomerulosclerosis. The exact mechanism of this effect is not fully understood. We examined the effects of transmural pressure alone on cell proliferation and DNA synthesis and investigated the role of platelet-derived growth factor (PDGF) and basic fibroblast growth factor (bFGF), candidates for mediation of glomerular diseases, in the pressure-induced events. Pressure was applied to cultured MC placed in a sealed chamber using compressed helium gas. Application of pressure resulted in a time-dependent (∼2 h) and pressure level-dependent (∼80 mmHg) increase in cell number (1.4-fold) and [3H]thymidine incorporation (2.7-fold). Pressure-induced DNA synthesis was significantly suppressed by inhibitors of phospholipase C (2-nitro-4-carboxyphenyl- N, N-diphenylcarbamate), protein kinase C [1-(5-isoquinolinylsulfonyl)-2-methylpiperazine and chelerythrine], or tyrosine kinases (genistein). Pressure caused a rapid but transient formation of inositol 1,4,5-trisphosphate, which was blocked by the phospholipase C inhibitor. Pressure also promoted a rapid increase in tyrosine kinase activity. Pressure increased mRNA levels of PDGF-B, with a peak at 6 h, but not those of PDGF-A or bFGF. Pressure-induced DNA synthesis was partially inhibited by a neutralizing anti-PDGF antibody but not by an antibody against bFGF or nonimmune IgG. Our results indicated that pressure by itself increases DNA synthesis and proliferation of cultured rat MC possibly through activation of protein kinase C and tyrosine kinases, and PDGF-B could be partially involved in these pathways.

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