Agonist Activation of δ-Opioid Receptor but not μ-Opioid Receptor Potentiates Fetal Calf Serum or Tyrosine Kinase Receptor-Mediated Cell Proliferation in a CellLine-Specific Manner

1997 ◽  
Vol 51 (1) ◽  
pp. 152-160 ◽  
Author(s):  
P. Y. Law ◽  
T. M. McGinn ◽  
K. M. Campbell ◽  
L. E. Erickson ◽  
H.H. Loh
Keyword(s):  
Cell Proliferation ◽  
Tyrosine Kinase ◽  
Opioid Receptor ◽  
Fetal Calf Serum ◽  
Calf Serum ◽  
Tyrosine Kinase Receptor ◽  
Specific Manner ◽  
Μ Opioid Receptor ◽  
Δ Opioid Receptor

scholarly journals Proteasome Involvement in Agonist-induced Down-regulation of μ and δ Opioid Receptors

2001 ◽  
Vol 276 (15) ◽  
pp. 12345-12355 ◽  
Author(s):  
Kirti Chaturvedi ◽  
Persis Bandari ◽  
Norihiro Chinen ◽  
Richard D. Howells
Keyword(s):  
Opioid Receptor ◽  
Opioid Receptors ◽  
Proteasome Inhibitors ◽  
Metabolic Labeling ◽  
Down Regulation ◽  
Hek 293 ◽  
293 Cells ◽  
Ubiquitin Proteasome ◽  
Μ Opioid Receptor ◽  
Δ Opioid Receptor

This study investigated the mechanism of agonist-induced opioid receptor down-regulation. Incubation of HEK 293 cells expressing FLAG-tagged δ and μ receptors with agonists caused a time-dependent decrease in opioid receptor levels assayed by immunoblotting. Pulse-chase experiments using [35S]methionine metabolic labeling indicated that the turnover rate of δ receptors was accelerated 5-fold following agonist stimulation. Inactivation of functional Giand Goproteins by pertussis toxin-attenuated down-regulation of the μ opioid receptor, while down-regulation of the δ opioid receptor was unaffected. Pretreatment of cells with inhibitors of lysosomal proteases, calpain, and caspases had little effect on μ and δ opioid receptor down-regulation. In marked contrast, pretreatment with proteasome inhibitors attenuated agonist-induced μ and δ receptor down-regulation. In addition, incubation of cells with proteasome inhibitors in the absence of agonists increased steady-state μ and δ opioid receptor levels. Immunoprecipitation of μ and δ opioid receptors followed by immunoblotting with ubiquitin antibodies suggested that preincubation with proteasome inhibitors promoted accumulation of polyubiquitinated receptors. These data provide evidence that the ubiquitin/proteasome pathway plays a role in agonist-induced down-regulation and basal turnover of opioid receptors.

Estrogen reduces proliferation and agonist-induced calcium increase in coronary artery smooth muscle cells

1997 ◽  
Vol 272 (4) ◽  
pp. H1996-H2003 ◽  
Author(s):  
R. C. Bhalla ◽  
K. F. Toth ◽  
R. A. Bhatty ◽  
L. P. Thompson ◽  
R. V. Sharma
Keyword(s):  
Cell Proliferation ◽  
Smooth Muscle ◽  
Coronary Artery ◽  
Guinea Pig ◽  
Untreated Control ◽  
Fetal Calf Serum ◽  
Thymidine Incorporation ◽  
Calf Serum ◽  
Causative Role ◽  
Pig Coronary Artery

Epidemiological evidence and estrogen replacement studies suggest that estrogen has a protective effect on the cardiovascular system against coronary artery disease. Vascular smooth muscle (VSM) cell replication has been shown to play a causative role in the pathogenesis of atherosclerosis. Therefore, in this study, we investigated the effect of chronic treatment of cultured guinea pig coronary artery VSM cells with physiological concentrations of 17beta-estradiol (E2) on thymidine incorporation, cell proliferation, and bradykinin-stimulated cytosolic calcium concentration ([Ca2+]i). Bradykinin at physiological concentrations causes contraction of endothelium-denuded guinea pig coronary artery rings in a concentration-dependent manner. VSM cells were first treated with low doses of E2 (10 pg/ml) for 1-2 days followed by treatment for 4-6 days with 50 pg/ml of E2, a concentration similar to that found in pregnancy. Using these protocols, we consistently observed the presence of E2-receptor mRNA in VSM cells by a ribonuclease protection assay. Fetal calf serum-stimulated [3H]thymidine incorporation was significantly reduced (P < 0.05) in E2-treated cells compared with untreated control cells. Similarly, E2 treatment significantly inhibited fetal calf serum-stimulated VSM cell proliferation compared with untreated control cells (P < 0.05). We also tested the hypothesis that E2 treatment attenuates agonist-stimulated [Ca2+]i in VSM cells because acute E2 treatment has been shown to produce relaxation of precontracted isolated coronary artery preparations. E2 treatment of VSM cells resulted in a significant decrease in bradykinin-stimulated [Ca2+]i compared with untreated cells (P < 0.05). In conclusion, our data demonstrate that estrogen at physiological concentrations directly regulates coronary VSM cell function.

Trastuzumab-HER/2 binding induced inhibition of cell growth and HER/2 tyrosine kinase activity

2007 ◽  
Vol 25 (18_suppl) ◽  
pp. 21146-21146
Author(s):  
S. Welt ◽  
T. Shay ◽  
C. Lanning ◽  
K. Horton ◽  
D. Kostyal
Keyword(s):  
Breast Cancer ◽  
Cell Growth ◽  
Tyrosine Kinase ◽  
Cell Lines ◽  
Fetal Calf Serum ◽  
Calf Serum ◽  
Breast Cancer Cell Lines ◽  
Inhibitory Effects ◽  
Her 2 ◽  
Post Menopausal

21146 Background: Cell lines containing the HER/2 amplicon are sensitive to Trastuzumab (T) mediated inhibition of cell growth. T blockade of cell growth signaling pathways have been hypothesized to occur by two mechanisms, inhibition of homo and hetro-dimerization of HER/2 with members of the EGFr family and inhibition of HER/2 phosphorylation. Methods: BT 474 and SK-BR3 breast cancer cell lines containing the HER/2 amplicon were cultured under condition demonstrating inhibitory effects of T by MTT and 3H-Thymidine incorporation (0.25–50 micrograms/ml for up to 9 days). RNA extracts from treated and un-treated cells were analyzed and compared using two immunoassays for phosphorylated HER/2. Various growth conditions were assessed for T inhibition of phosphorylation including, serum free media, fetal calf serum, human pre and post menopausal serum, breast cancer patient's serum and normal male serum. Results: Breast cancer cell lines BT 474 and SK-BR3 both demonstrated cell growth inhibition when cultured with T over a wide range of concentrations down to 0.25 microgram/ml. The inhibitory effects on SK-BR3 were low but statistically significant in both MTT and 3H-Thymidine incorporation assays. Examining this same concentration range of T on these cell lines demonstrated no appreciable reduction of phosphorylation by western blot analysis at various time points after exposure to T. Two different immune assays for HER/2 phosphorylation were analyzed. Further examination of replacement of fetal calf serum by human female pre and post menopausal serum samples or similar samples from breast cancer patients did not result in reduction of the phosphorylation signal. Conclusions: In this in vitro model T does not mediate effects through HER/2 tyrosine kinase inhibition under conditions that simulate clinical conditions. Whether inhibition of phosphorylation will be apparent at later time points is unknown. These finding may explain the activity of lapatinib, a HER/2 tyrosine kinase inhibitor, on T resistant breast cancer. No significant financial relationships to disclose.

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