The purinergic component of human bladder smooth muscle cells' proliferation and contraction under physiological stretch

Objective: To investigate whether cyclic stretch induces proliferation and contraction of human smooth muscle cells (HBSMCs), mediated by P2X purinoceptor 1 and 2 and the signal transduction mechanisms of this process. Methods: HBSMCs were seeded on silicone membrane and stretched under varying parameters; (equibiaxial elongation: 2.5%, 5%, 10%, 15%, 20%, 25%), (Frequency: 0.05 Hz, 0.1 Hz, 0.2 Hz, 0.5 Hz, 1 Hz). 5-Bromo-2-deoxyuridine assay was employed for proliferative studies. Contractility of the cells was determined using collagen gel contraction assay. After optimal physiological stretch was established; P2X1 and P2X2 were analyzed by real time polymerase chain reaction and Western Blot. Specificity of purinoceptors was maintained by employing specific inhibitors; (NF023 for P2X1, and A317491for P2X2), in some experiments. Results: Optimum proliferation and contractility were observed at 5% and 10% equibiaxial stretching respectively, applied at a frequency of 0.1 Hz; At 5% stretch, proliferation increased from 0.837 +/- 0.026 (control) to 1.462 +/- 0.023%, p<0.05. Mean contraction at 10% stretching increased from 31.7 +/- 2.3%, (control) to 78.28 +/- 1.45%, p< 0.05. Expression of P2X1 and P2X2 was upregulated after application of stretch. Inhibition had effects on proliferation (1.232 +/- 0.051, p<0.05 NF023) and (1.302 +/- 0.021, p<0.05 A314791) while contractility was markedly reduced (68.24 +/- 2.31, p<0.05 NF023) and (73.2 +/- 2.87, p<0.05 A314791). These findings shows that mechanical stretch can promote magnitude-dependent proliferative and contractile modulation of HBSMCs in vitro, and P2X1 and 2 are at least partially responsible in this process. (C) 2013 Elsevier Inc. All rights reserved.