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The effects of inhibiting endocytosis on 5HT-mediated proliferation and ERK activation 5HT is known to have mitogenic effects on cells of the pulmonary artery (Welsh et al., 2004). It has previously been shown that 5HT induced proliferation is mediated via the extracellular signal-regulated kinase (ERK) pathway (Lee et al., 1999). Furthermore, it has been found that in some instances receptor-mediated endocytosis is required for ERK activation (Della Rocca et al., 1999). The Chinese hamster lung fibroblast cell line CCL39 was employed to investigate if both 5HT receptors and the 5HT transporter (5HTT) are involved in 5HT-induced ERK activation and to determine what role endocytosis plays in this process. To monitor the effects of 5HT (0.01 μM to 10 μM) on ERK activation, immunoblotting using a phospho-specific ERK antibody was undertaken. Structurally unrelated compounds monodansylcadaverin (MDC) (100 μM) and conconavilin A (ConA) (0.25mg/ml) were administered to inhibit endocytosis. 5HTT mediated responses were assessed using 5HTT inhibitors citalopram (1 μM) and fluoxetine (10 μM). Conversely, blockade of 5HT receptors using methiothepin (5HT1/2, 1 μM) or ketanserin (5HT2A, 10 μM) and GR55562 (5HT1B/1D, 10 m M) was investigated. Proliferation was determined by monitoring [3H]-thymidine incorporation. Statistical analysis was by students unpaired t-test or by one-way analysis of variance with Tukey post test where appropriate. Results expressed as mean ± S.E.M. Blocking ERK activity with MEK inhibitor U0126 (1μM) resulted in a reduction of proliferation to 30 ± 9% (n = 4, p<0.01) of the maximal 5HT effect. Inhibition of all 5HT1/2 receptors using methiothepin decreased maximal ERK activation by 50 ± 7% (n=3, p<0.05). Simultaneous inhibition of 5HT1B and 5HT2A receptors also attenuated ERK activation, reducing it by 20.6 ± 4% at 0.1 μM 5HT and by 37.8 ± 4% at 5HT 1 μM (n=3 p<0.01). Inhibition of 5HTT with either fluoxetine or citalopram also decreased ERK, with citalopram inhibiting the maximal response by 34% ± 9% (n=3, p<0.05) and fluoxetine inhibiting responses to 0.1 μM and 1 μM 5HT (27.7 ± 1.7% and 28.9 ± 6.3% respectively n=3, p<0.05). MDC had no effect on receptor mediated ERK activation (Control: 100% n=3; MDC: 95.7 ± 6.5% n=3), with similar results obtained using conA (Control: 100% n=3; ConA 99.4± 15.3 % n=3). Inhibition of endocytosis using either MDC or conA was unable to alter 5HTT mediated ERK activation (Control: 100 ± 0.4 % n=3; MDC: 105.6 ± 8.2% n=3; ConA: 80.8 ± 6.8% n=3). In summary, 5HT induces proliferation of CCL-39 cells, a process that requires intracellular activation of ERK. While 5HT receptors and 5HTT both appear to be involved in this signalling pathway, endocytosis appears not to be a requirement for 5HT to activate ERK via neither 5HT receptors nor 5HTT.
Della Rocca et al., (1999) J Biol Chem 274: 4749-4753 |
