Body fluid balance is controlled by alteration of urine concentration, under the influence of vasopressin (VP). VP, via cAMP and protein kinase A (PKA), causes exocytosis of AQP2 water channels in the collecting duct (Nielsen et al., 1995). Local factors such as bradykinin (BK) may modulate this response. BK probably acts via B₂ receptors, coupled to G_i or G_q, either inactivating adenylate cyclase, or activating protein kinase C (PKC) respectively. This study aims to determine the effects of BK on AQP2 shuttling, and to elucidate the second messengers involved. An understanding of these mechanisms may improve management of water balance disorders. Male Wistar rats were humanely killed by terminal anaesthesia with pentobarbitone sodium (240mg/kg I.P.) and cervical dislocation. The kidneys were removed and inner medullary tubules prepared as previously described (Shaw & Marples, 2002). The tubule suspension was divided into four aliquots treated as follows: control, VP (1nM), BK (10nM), VP + BK. The tubules then either underwent fractionation into plasma membrane (PM) and intracellular vesicle (ICV)-enriched fractions to determine the cellular AQP2 distribution by western blotting (expressed as a PM:ICV ratio normalised to the control), or were lysed in 0.1M HCl to release cytosolic cAMP, which was quantified using a standard kit (Sigma). Total protein from these samples was western blotted using a phospho-specific AQP2 antibody. Data are presented as means ± S.E. and compared using the false discovery rate procedure (Curran-Everett, 2000). VP stimulation caused a shift of AQP2 from ICV to the PM. In contrast, BK caused a significant decrease in the PM:ICV ratio, indicating either a decrease in constitutive AQP2 shuttling, or an increase in AQP2 endocytosis. When VP and BK were added simultaneously, the increase in PM:ICV ratio previously seen with VP was no longer evident, demonstrating that BK completely inhibits the VP induced shuttling of AQP2. As expected, VP caused an increase in cAMP and AQP2 phosphorylation, but neither effect was inhibited by BK. BK also had no effect on basal cAMP and AQP2 phosphorylation levels. In summary, BK reduces basal AQP2 in the plasma membrane, and inhibits AQP2 shuttling distal to AQP2 phosphorylation. These effects are consistent with the known diuretic effect of BK and may contribute to the hypotensive properties of ACE inhibitors.