Effect of lipopolysaccharide from escherichia coli on urothelial stretch induced ATP release

Introduction. Urinary tract infection (UTI) is experienced by 50% of all women at least once during their lifetime, commonly caused by Escherichia coli. The symptoms of UTI include increased voiding frequency and urgency, usually accompanied by a sensation of burning pain and foul smelling urine. ATP released from bladder urothelium is an important molecule for signalling urinary urgency. Aims. The aim of the current study was to examine the effect of lipopolysaccharide (LPS) from E. coli on stretchinduced ATP release from urothelial RT4 cells in culture. Methods. Urothelial RT4 cells were cultured in 24-well plates in McCoys 5A culture media (with foetal bovine serum, glutamate and antibiotics) at 37oC with 5%CO2. At confluence cells were treated with (10μg/mL) E. coli LPS. Two different LPS serotypes were used, O111 from non-pthogenic E. coli and O55 from enteropathogenic E. coli. After 24h cells were incubated for 10min with Krebs as control or hypotonic Krebs solution (50%) to mimic stretch. Supernatant was collected and the ATP concentration measured (Bioluminescence Assay). Results are presented as nM ATP released [median (IQR)]. Cells pre-treated with different LPS serotypes are compared using a non-parametric Kruskal-Wallis test. Results. Basal ATP release was unaffected by LPS pre-treatment [control 14.2nM (7.2-20.7); serotype O111 16.7nM (8.2-27.9); serotype O55 12.9 (7.9-17.6), n=8, P=0.88]. Hypotonic media induced an increase in ATP release [48.75nM (12.4-51.7), n=8]. This was not affected by pre-treatment with LPS serotype O111 [35.43nM (11.2-65.6), n=8]. Pre-treatment with LPS serotype O55 significantly reduced stretch induced ATP release [10.6nM (3.1-14.2), n=8, P=0.025]. Discussion. The decrease in stretch-induced ATP release by pathogenic (O55) but not by non-pathogenic (O111) E. coli LPS suggests that infection may alter urothelial purinergic signaling in response to filling