Stacey Barrick

Altered urinary bladder function in mice lacking the vanilloid receptor TRPV1

In the urinary bladder, the capsaicin-gated ion channel TRPV1 is expressed both within afferent nerve terminals and within the epithelial cells that line the bladder lumen. To determine the significance of this expression pattern, we analyzed bladder function in mice lacking TRPV1. Compared with wild-type littermates, trpv1−/− mice had a higher frequency of low-amplitude, non-voiding bladder contractions. This alteration was accompanied by reductions in both spinal cord signaling and reflex voiding during bladder filling (under anesthesia). In vitro, stretch-evoked ATP release and membrane capacitance changes were diminished in bladders excised from trpv1−/− mice, as was hypoosmolality-evoked ATP release from cultured trpv1−/− urothelial cells. These findings indicate that TRPV1 participates in normal bladder function and is essential for normal mechanically evoked purinergic signaling by the urothelium.

ATP and purinergic receptor–dependent membrane traffic in bladder umbrella cells

The umbrella cells that line the bladder are mechanosensitive, and bladder filling increases the apical surface area of these cells; however, the upstream signals that regulate this process are unknown. Increased pressure stimulated ATP release from the isolated uroepithelium of rabbit bladders, which was blocked by inhibitors of vesicular transport, connexin hemichannels, ABC protein family members, and nucleoside transporters. Pressure-induced increases in membrane capacitance (a measure of apical plasma membrane surface area where 1 microF approximately equals 1 cm2) were inhibited by the serosal, but not mucosal, addition of apyrase or the purinergic receptor antagonist PPADS. Upon addition of purinergic receptor agonists, increased capacitance was observed even in the absence of pressure. Moreover, knockout mice lacking expression of P2X2 and/or P2X3 receptors failed to show increases in apical surface area when exposed to hydrostatic pressure. Treatments that prevented release of Ca2+ from intracellular stores or activation of PKA blocked ATPgammaS-stimulated changes in capacitance. These results indicate that increased hydrostatic pressure stimulates release of ATP from the uroepithelium and that upon binding to P2X and possibly P2Y receptors on the umbrella cell, downstream Ca2+ and PKA second messenger cascades may act to stimulate membrane insertion at the apical pole of these cells.

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

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 (EC50= 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 [Ca2+]i in CYP (24 h)‐treated cultured rat urothelial cells with a selective B1 receptor agonist (des‐Arg9‐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‐Arg10‐Hoe‐140) or B2 (Hoe‐140) receptor antagonists. These studies demonstrate that urothelial expression of bradykinin receptors is plastic and is altered by pathology.

Activation of Urothelial Transient Receptor Potential Vanilloid 4 by 4α-Phorbol 12,13-Didecanoate Contributes to Altered Bladder Reflexes in the Rat

The ion channel transient receptor potential vanilloid (TRPV) 4 can be activated by hypo-osmolarity, heat, or certain lipid compounds. Here, we demonstrate expression of functional TRPV4 protein in the urothelium lining the renal pelvis, ureters, urinary bladder, and urethra. Exposure of cultured rat urothelial cells from the urinary bladder to the TRPV4-selective agonist 4α-phorbol 12,13-didecanoate (4α-PDD) promoted Ca2+ influx, evoked ATP release, and augmented the ATP release evoked by hypo-osmolarity. In awake rats during continuous infusion cystometrograms, intravesical administration of 4α-PDD (10–100 μM) increased maximal micturition pressure by 51%, specifically by augmenting the portion of each intravesical pressure wave that follows high-frequency urethral oscillations and voiding. This unusual pharmacological effect was prevented by intravesical pretreatment with the nonselective ATP receptor antagonist, pyridoxal phosphate-6-azophenyl-2′,4′-disulfonic acid (100 μM), systemic treatment with the selective P2X3 purinergic antagonist 5-([(3-phenoxybenzyl)[1S)-1,2,3,4-tetrahydro-1-naphthalenyl]amino]carbonyl)-1,2,4-benzenetricarboxylic acid (A317491) (250 μmol/kg), or urethane anesthesia, but was unaffected by capsaicin pretreatment (100 mg/kg s.c.) or denervation of the urethral sphincter. 4α-PDD (1–100 μM) did not alter the contractility to electrical stimulation of excised bladder strips. We conclude that activation of urothelial TRPV4 by 4α-PDD and release of mediators such as ATP trigger a novel neural mechanism that regulates the late phase of detrusor muscle contraction after micturition. These data raise the possibility that TRPV4 channels in the urothelium could contribute to abnormal bladder activity.

Heterogeneity of muscarinic receptor-mediated Ca2+ responses in cultured urothelial cells from rat.

Muscarinic receptors (mAChRs) have been identified in the urothelium, a tissue that may be involved in bladder sensory mechanisms. This study investigates the expression and function of mAChRs using cultured urothelial cells from the rat. RT-PCR established the expression of all five mAChR subtypes. Muscarinic agonists acetylcholine (ACh; 10 microM), muscarine (Musc; 20 microM), and oxotremorine methiodide (OxoM; 0.001-20 microM) elicited transient repeatable increases in the intracellular calcium concentration ([Ca(2+)](i)) in approximately 50% of cells. These effects were blocked by the mAChR antagonist atropine methyl nitrate (10 microM). The sources of [Ca(2+)](i) changes included influx from external milieu in 63% of cells and influx from external milieu plus release from internal stores in 27% of cells. The use of specific agonists and antagonists (10 microM M(1) agonist McN-A-343; 10 microM M(2), M(3) antagonists AF-DX 116, 4-DAMP) revealed that M(1), M(2), M(3) subtypes were involved in [Ca(2+)](i) changes. The PLC inhibitor U-73122 (10 microM) abolished OxoM-elicited Ca(2+) responses in the presence of the M(2) antagonist AF-DX 116, suggesting that M(1), M(3), or M(5) mediates [Ca(2+)](i) increases via PLC pathway. ACh (0.1 microM), Musc (10 microM), oxotremorine sesquifumarate (20 microM), and McN-A-343 (1 muM) acting on M(1), M(2), and M(3) mAChR subtypes stimulated ATP release from cultured urothelial cells. In summary, cultured urothelial cells express functional M(1), M(2), and M(3) mAChR subtypes whose activation results in ATP release, possibly through mechanisms involving [Ca(2+)](i) changes.

A Potent α/β-Peptide Analogue of GLP-1 with Prolonged Action in Vivo

Glucagon-like peptide-1 (GLP-1) is a natural agonist for GLP-1R, a G protein-coupled receptor (GPCR) on the surface of pancreatic β cells. GLP-1R agoinsts are attractive for treatment of type 2 diabetes, but GLP-1 itself is rapidly degraded by peptidases in vivo. We describe a design strategy for retaining GLP-1-like activity while engendering prolonged activity in vivo, based on strategic replacement of native α residues with conformationally constrained β-amino acid residues. This backbone-modification approach may be useful for developing stabilized analogues of other peptide hormones.

Effect of botulinum toxin A on urothelial-release of ATP and expression of SNARE targets within the urothelium

Botulinum neurotoxin serotype A (BoNT/A) has emerged as an effective treatment of urinary bladder overactivity. Intravesical lipotoxin (BoNT/A delivery using liposomes), which may target the urothelium, is effective in blocking acetic acid induced hyperactivity in animals. The objective of this study was to assess the possible site of toxin action within the urothelium.

Direct interaction between NHERF1 and Frizzled regulates β-catenin signaling

Although Wnt-Frizzled (Fzd) signaling is critical in the pathophysiology of carcinomas, its role in human breast cancer has been difficult to establish. We show here that the adaptor protein Na+/H+ exchange regulatory factor1 (NHERF1), a protein abundantly expressed in normal mammary epithelium, regulates Wnt signaling, maintaining low levels of β-catenin activation. NHERF1s effects are mediated by direct interactions between one of its PSD-95/drosophila discs large/ZO-1 (PDZ) domains and the C-terminus of a subset of Fzd receptors. Loss of NHERF1 in breast cancer cell lines enhances canonical Wnt signaling and Wnt-dependent cell proliferation. Furthermore, the mammary glands of NHERF1-knockout mice exhibit increased mammary duct density accompanied by increased proliferation and β-catenin activity. Finally, we demonstrate a negative correlation between NHERF1 expression and nuclear β-catenin in human breast carcinomas. Taken together, these results provide a novel insight into the regulation of Wnt signaling in normal and neoplastic breast tissues, and identify NHERF1 as an important regulator of the pathogenesis of breast tumors.

EBP50 inhibits the anti-mitogenic action of the parathyroid hormone type 1 receptor in vascular smooth muscle cells

Parathyroid hormone-related protein (PTHrP) and the parathyroid hormone type 1 receptor (PTH1R) are important regulators of vascular remodeling. PTHrP expression is associated to increased proliferation of vascular smooth muscle cells (VSMC). In contrast, signaling via the PTH1R inhibits cell growth. The mechanisms regulating the dual effect of PTHrP and PTH1R on VSMC proliferation are only partially understood. In this study we examined the role of the adaptor protein ezrin-radixin-moesin-binding phosphoprotein (EBP50) on PTH1R expression, trafficking, signaling and control of A10 cell proliferation. In normal rat vascular tissues, EBP50 was restricted to the endothelium with little expression in VSMC. EBP50 expression significantly increased in VSMC following angioplasty in parallel with PTHrP. Interestingly, PTHrP was able to induce EBP50 expression. In the clonal rat aortic smooth muscle cell line A10, EBP50 increased the recruitment of PTH1R to the cell membrane and delayed its internalization in response to PTHrP(1-36). This effect required an intact C-terminal motif in the PTH1R. In naïve A10 cells, PTHrP(1-36) stimulated cAMP production but not intracellular calcium release. In contrast, PTHrP(1-36) induced both cAMP and calcium signaling in A10 cells over-expressing EBP50. Finally, EBP50 attenuated the induction of p27(kip1) and the anti-proliferative effect of PTHrP(1-36). In summary, this study demonstrates the dynamic expression of EBP50 in vessels following injury and the effects of EBP50 on PTH1R function in VSMC. These findings highlight one of the mechanisms leading to increased VSMC proliferation and have important implication in the understanding of the molecular events leading to restenosis.

PEX7 and EBP50 target iNOS to the peroxisome in hepatocytes.

iNOS localizes to both the cytosol and peroxisomes in hepatocytes in vitro and in vivo. The structural determinants for iNOS localization are not known. One plausible mechanism for iNOS localization to the peroxisome is through the interaction with peroxisomal import proteins PEX5 or PEX7. siRNA knockdown of PEX7 reduced iNOS colocalization with the peroxisomal protein PMP70. Proteomic studies using MALDI-MS identified iNOS association with the 50-kD ezrin binding PDZ protein (EBP50). Confocal microscopy studies and immunoelectron microscopy confirmed iNOS association with EBP50, with greatest colocalization occurring at 8h of cytokine exposure. EBP50 associated with peroxisomes in a PEX5 and PEX7-dependent manner. iNOS localization to peroxisomes was contingent on EBP50 expression in LPS-treated mice. Thus, iNOS targeting to peroxisomes in hepatocytes involves interaction with PEX7 and EBP50. The targeting of iNOS protein to the peroxisome may shift the balance of metabolic processes that rely on heme proteins susceptible to modification by radical oxygen and nitrogen radicals.