Proliferation of the human urothelium is induced by atypical β1-adrenoceptors

1. We wanted to assess whether β-adrenoceptors mediate proliferation in the normal and malignant urothelial cell lines UROtsa and T24, respectively.
2. Urothelial cells were cultured for 24 h in the presence of the β-adrenoceptor agonists isoprenaline (β_1/2/3), dobutamine (β₁), salbutamol (β₂), BRL 37344 (β₃), CGP 12177 (a partial β-agonist) or β-adrenoceptor antagonists (metoprolol; β₁, propranolol; β_1/2). Phosphorylation of kinases was screened with a Human Phospho-Kinase Array Kit (R&D systems). Intracellular pathways activated by proliferation of urothelial cells were characterized by incubating cells with the MEK1/2 inhibitor PD 98,059, the p38 kinase inhibitor losmapimod or with the Akt 1/2 kinase inhibitor. Proliferation was assessed with the MTT proliferation assay (ATCC). Western blot and immunocytochemistry were used for detection of the β₁-adrenoceptor.
3. Isoprenaline and dobutamine induced proliferation, while salbutamol and BRL 37344 did not. Dobutamine-induced proliferation was not affected by metoprolol or propranolol but was instead antagonized by CGP 12177 in T24 but not in UROtsa. In response to stimulation with dobutamine, Akt1/2/3 was phosphorylated in UROtsa, while ERK1/2 and p38 were phosphorylated in T24. MEK1/2 inhibition blocked basal and dobutamine-induced proliferation in T24 but only basal proliferation in UROtsa. Losmapimod slightly inhibited basal proliferation in T24 but not dobutamine-induced proliferation. Akt 1/2 inhibitor blocked basal and dobutamine-induced proliferation in UROtsa. Immunocytochemistry and Western blot revealed expression of β₁-adrenoceptors in both urothelial cell lines.
4. The present data show that the urothelium expresses atypical β1-adrenoceptors that activate intracellular kinases inducing urothelial proliferation.