Figure 3.

Adrenergic receptors do not modulate cardiomyocyte NGF secretion. Dissociated cardiomyocytes were cultured for 3 d before treatment with phenylephrine or isoproterenol. A, Cardiomyocytes in culture show typical actin cytoskeleton after staining with phalloidin. B, C, Cardiomyocytes express NGF protein (B) and the pro-neurotrophin convertase furin (C). Scale bars, 50 μm. D, Staining of Western blots loaded with different amounts of recombinant human β-NGF showed that antibody sensitivity is at least 10 ng/lane. E, After culture for 48 h, Western blot analysis of the culture medium revealed immunoreactive NGF, mainly as pro-NGF with a molecular mass of ∼40 kDa (left) and preincubation of the antibody with an NGF-blocking peptide resulted in absence NGF detection (right). F, Probing with an antibody recognizing a non-NGF epitope in the pro region of the protein confirmed that the 40 kDa band is pro-NGF. G, Treatment of the cardiomyocyte cultures with 0.1–10 μm concentrations of the α-agonist phenylephrine (PE) or the β-agonist isoproterenol (IS) did not alter NGF content within the medium.