Expression and function of bradykinin B1 and B2 receptors in normal and inflamed rat urinary bladder urothelium

Abstract

The bladder urothelium exhibits dynamic sensory properties that adapt to changes in the local environment. These studies investigated the localization and function of bradykinin receptor subtypes B1 and B2 in the normal and inflamed (cyclophosphamide (CYP)-induced cystitis) bladder urothelium and their contribution to lower urinary tract function in the rat. Our findings indicate that the bradykinin 2 receptor (B2R) but not the bradykinin 1 receptor (B1R) is expressed in control bladder urothelium. B2R immunoreactivity was localized throughout the bladder, including the urothelium and detrusor smooth muscle. Bradykinin-evoked activation of this receptor elevated intracellular calcium (EC(50) = 8.4 nM) in a concentration-related manner and evoked ATP release from control cultured rat urothelial cells. In contrast, B1R mRNA was not detected in control rat urinary bladder; however, following acute (24 h) and chronic (8 day) CYP-induced cystitis in the rat, B1R mRNA was detected throughout the bladder. Functional B1Rs were demonstrated by evoking ATP release and increases in [Ca(2+)](i) in CYP (24 h)-treated cultured rat urothelial cells with a selective B1 receptor agonist (des-Arg(9)-bradykinin). Cystometry performed on control anaesthetized rats revealed that intravesical instillation of bradykinin activated the micturition pathway. Attenuation of this response by the P2 receptor antagonist PPADS suggests that bradykinin-induced micturition facilitation may be due in part to increased purinergic responsiveness. CYP (24 h)-treated rats demonstrated bladder hyperactivity that was significantly reduced by intravesical administration of either B1 (des-Arg(10)-Hoe-140) or B2 (Hoe-140) receptor antagonists. These studies demonstrate that urothelial expression of bradykinin receptors is plastic and is altered by pathology.

Figures

Figure 1. Bradykinin B1 and B2 receptor mRNA expression in the rat urinary bladder

A, time course of changes in bradykinin B1 and B2 receptor mRNA in the urinary bladder detrusor smooth muscle and urothelium in normal and cyclophosphamide (CYP)-treated rats (24 h and 8 day (8 d) treatments). GAPDH was used as a control. B, relative product strengths of B1 and B2 receptor mRNA determined by densitometry (density of product/density of GAPDH) in normal and CYP-treated rats. ND = none detected.

Figure 2. Bradykinin B2 receptor immunocytochemistry in the rat urinary bladder

A, bradykinin B2 receptor expression was distributed in the rat urinary bladder, with staining evident in the urothelium (white arrows). B, B2 receptor expression was present in both the underlying basal and apical cells of the urothelium (white arrows) and detrusor smooth muscle (asterisk). C, B2 receptor immunoreactivity was present predominantly on the plasma membrane and cytoplasmically. D, preabsorption of the B2 receptor antibody with the C-terminal antigenic peptide reduced staining to background levels.

Figure 3. Bradykinin-evoked changes in [Ca2+]i in control cultured rat urothelial cells

A, discontinuous representative traces of changes in F340/F380 in cultured rat urothelial cells following bath application of varying concentrations of bradykinin (1–100 nm) and ATP (10 μm). B, concentration–response curve for bradykinin-mediated changes in F340/F380 in cultured rat urothelial cells. Data are expressed as mean (± s.e.m.) change in F340/F380 as a percentage of the maximum ATP (10 μm) response.

Figure 4. Bradykinin evoked changes in [Ca2+]i in control and CYP (24 h)-treated cultured rat urothelial cells

A, bradykinin (30 nm) consistently and reproducibly evoked an increase in [Ca2+]i in cultured rat urothelial cells. B, bradykinin (30 nm)-evoked increase in [Ca2+]i was significantly attenuated in the presence of the B2 selective antagonist, Hoe-140 (1 μm). C, the B1 selective agonist, des-Arg9-bradykinin (des-Arg9-BK; 10 μm) had a significantly smaller effect on [Ca2+]i in control rat urothelial cells compared to bradykinin (30 nm) alone. D, following CYP (24 h)-treatment, however, populations of cultured urothelial cells were responsive to des-Arg9-bradykinin (10 μm). **P≤ 0.01.

Figure 5. Bradykinin evoked the release of ATP from rat urothelial cells

A, representative time course recordings of ATP release evoked from separate control cultured rat urothelial cells following either simulation with bradykinin (30 nm, BK), bradykinin (30 nm) in the presence of Hoe-140 (1 μm), des-Arg9-bradykinin (10 μm, des-Arg9-BK) and the effect of des-Arg9-bradykinin (10 μm) in the presence of Hoe-140 (1 μm) on urothelial cells cultured from CYP (24 h)-treated rats. All agonists were applied at time = 0. Cells were pretreated with antagonists 10 min prior to agonist application. B, histograms illustrating mean release of ATP from cultured rat urothelial cells following stimulation as described above. **P≤ 0.01.

Figure 6. Transepithelial resistance (TER) and permeabilities of normal and CYP (24 h)-treated rat urothelium

A, bradykinin B2 receptor expression was assessed in urinary bladder of CYP (24 h)-treated rats. In contrast to control urinary bladder (Fig. 2A), CYP-treated bladder urothelium exhibited a loss of compact urothelial structure (arrows). B, transepithelial resistance in CYP-treated bladders was significantly reduced compared to control. **P≤ 0.01.

Figure 7. Bradykinin facilitates the micturition reflex in part by increased purinergic responsiveness

Representative infusion cystometric recordings obtained from (separate) anaesthetized rats, illustrating urinary bladder contractions following infusion of physiological saline (A; 0.04 ml min−1) and then followed by infusion of bradykinin (B; 100 μm). In a separate series of experiments, bradykinin (100 μm) was infused in the presence of the B2 receptor antagonist Hoe-140 (C; 10 μm) or in the presence of the non-selective P2 receptor antagonist PPADS (D; 10 mm). E and F, bradykinin (BK) facilitates the micturition reflex as indicated by a significant decrease in intercontractile interval (ICI) between voiding episodes (n = 4, **P < 0.01) and increased bladder contraction amplitude (BCA; n = 4, *P = 0.03). These changes in urodynamic function were significantly attenuated following pretreatment with the B2 receptor antagonist Hoe-140 (10 μm) or the P2 purinergic receptor antagonist PPADS (10 mm).

Figure 8. Cyclophosphamide (24 h)-induced bladder hyperreflexia involves B1 and B2 receptors

A, rats pretreated with CYP (24 h) displayed bladder hyperreflexia as indicated by a significant decrease in ICI and increase in non-voiding contractions (NVCs). B, following instillation of B1 receptor antagonist, des-Arg10-Hoe-140 (100 nm), a significant attenuation of the frequency of NVCs was observable. C, discontinuous recording from a rat (CYP (24 h)-treated) prior to administration of Hoe-140 (10 μm). D, Hoe-140 (10 μm) had no significant effect on the frequency of NVCs but did significantly increase the ICI of CYP-treated rats.