Bradykinin modulates spontaneous nerve growth factor production and stretch-induced ATP release in human urothelium

The urothelium plays a crucial role in integrating urinary bladder sensory outputs, responding to mechanical stress and chemical stimulation by producing several diffusible mediators, including ATP and, possibly, neurotrophin nerve growth factor (NGF). Such urothelial mediators activate underlying afferents and thus may contribute to normal bladder sensation and possibly to the development of bladder overactivity. The muscle-contracting and pain-inducing peptide bradykinin is produced in various inflammatory and non-inflammatory pathologies associated with bladder overactivity, but the effect of bradykinin on human urothelial function has not yet been characterized. The human urothelial cell line UROtsa expresses mRNA for both B1 and B2 subtypes of bradykinin receptors, as determined by real-time PCR. Bradykinin concentration-dependently (pEC50=8.3, Emax 4434±277nM) increased urothelial intracellular calcium levels and induced phosphorylation of the mitogen-activated protein kinase (MAPK) ERK1/2. Activation of both bradykinin-induced signaling pathways was completely abolished by the B2 antagonist icatibant (1μM), but not the B1 antagonist R715 (1μM). Bradykinin-induced (100nM) B2 receptor activation markedly increased (192±13% of control levels) stretch-induced ATP release from UROtsa in hypotonic medium, the effect being dependent on intracellular calcium elevations. UROtsa cells also expressed mRNA and protein for NGF and spontaneously released NGF to the medium in the course of hours (11.5±1.4pgNGF/mgprotein/h). Bradykinin increased NGF mRNA expression and accelerated urothelial NGF release to 127±5% in a protein kinase C- and ERK1/2-dependent manner. Finally, bradykinin up-regulated mRNA for transient-receptor potential vanilloid (TRPV1) sensory ion channel in UROtsa. In conclusion, we show that bradykinin represents a versatile modulator of human urothelial phenotype, accelerating stretch-induced ATP release, spontaneous release of NGF, as well as expression of sensory ion channel TRPV1. Bradykinin-induced changes in urothelial sensory function might contribute to the development of bladder dysfunction.