Analysis of chronic Morphine-induced changes in ErbB signalling pathways.

<p>(<b>A</b>) Effect of protein kinase blockers on basal and Heregulin-stimulated ERK1/2 and Akt phosphorylation. BT474 cells were cultured for 5 d in the absence (control) or presence of Morphine (10 µM), before the impact of ErbB1 (AG1478), ErbB2 (AG825), PI3K (Wortmannin; Wort.) and metalloproteinases (EGCG) on basal and Heregulin (40 ng/ml)-stimulated ERK1/2 and Akt signalling was determined by Western blot using phospho-specific antibodies. Controls were kept in the absence of protein inhibitors. To verify equal protein loading, controls were also stained with overall reactive anti-ERK1/2 and anti-Akt antibodies. (<b>B</b>) Involvement of ErbB2 on basal and EGF-stimulated ERK1/2 and Akt signalling. Controls and cells chronically exposed to Morphine (10 µM; 5d) were incubated with or without AG825 (50 µM; 30 min), before ERK1/2 and Akt phosphorylation was determined for 5 min in the absence or presence of EGF (100 ng/ml). Equal protein loading was verified by staining controls with overall reactive anti-ERK1/2 and anti-Akt antibodies. (<b>C</b>) Regulation of ErbB receptor abundance by chronic Morphine treatment in BT474 cells. Cells were cultured for 5 d in the presence or absence of Morphine (10 µM) and Naloxone (100 µM) as indicated and overall ErbB receptor levels were analysed by Western blot using specific antibodies for ErbB1 (175 kDa), ErbB2 (185 kDa), ErbB3 (185 kDa) and ErbB4 (170 kDa). Equal protein loading was verified by incubation of the blots with an antibody against β-tubulin. (<b>D</b>) Alteration of ErbB1 containing receptor dimers by chronic Morphine. Controls and chronically Morphine (10 µM; 5 d)-treated BT474 cells were stimulated for 5 min with or without Heregulin (40 ng/ml) to form receptor dimers. Proteins were cross-linked and ErbB1 containing dimers were immunoprecipitated using an anti-ErbB1 antibody. Individual ErbB receptors were determined in whole cell solubilisates (S) and immunoprecipitates (IP) by Western blot using type-specific antibodies. Equal protein load was verified by determination of IgG heavy chains (<i>F_c</i>).