RESUMEN
Venezuelan equine encephalitis virus (VEEV) is a positively-stranded RNA arbovirus of the genus Alphavirus that causes encephalitis in humans. Cynomolgus macaques are a relevant model of the human disease caused by VEEV and are useful in exploring pathogenic mechanisms and the host response to VEEV infection. Macaques were exposed to small-particle aerosols containing virus derived from an infectious clone of VEEV strain INH-9813, a subtype IC strain isolated from a human infection. VEEV-exposed macaques developed a biphasic fever after infection similar to that seen in humans. Maximum temperature deviation correlated with the inhaled dose, but fever duration did not. Neurological signs, suggestive of virus penetration into the central nervous system (CNS), were predominantly seen in the second febrile period. Electroencephalography data indicated a statistically significant decrease in all power bands and circadian index during the second febrile period that returned to normal after fever resolved. Intracranial pressure increased late in the second febrile period. On day 6 post-infection macaques had high levels of MCP-1 and IP-10 chemokines in the CNS, as well as a marked increase of T lymphocytes and activated microglia. More than four weeks after infection, VEEV genomic RNA was found in the brain, cerebrospinal fluid and cervical lymph nodes. Pro-inflammatory cytokines & chemokines, infiltrating leukocytes and pathological changes were seen in the CNS tissues of macaques euthanized at these times. These data are consistent with persistence of virus replication and/or genomic RNA and potentially, inflammatory sequelae in the central nervous system after resolution of acute VEEV disease.
Asunto(s)
Virus de la Encefalitis Equina Venezolana , Encefalomielitis Equina Venezolana , Animales , Sistema Nervioso Central , Virus de la Encefalitis Equina Venezolana/genética , Caballos/genética , Inflamación , Macaca fascicularis , ARN Viral/genéticaRESUMEN
The regulation of the cell cycle by the ubiquitin-proteasome system is dependent on the activity of E3 ligases. Skp2 (S-phase kinase associated protein-2) is the substrate recognition subunit of the E3 ligase that ubiquitylates the cell cycle inhibitors p21(cip1) and p27(kip1) thus promoting cell cycle progression. Increased expression of Skp2 is frequently observed in diseases characterized by excessive cell proliferation, such as cancer and neointima hyperplasia. The stability and cellular localization of Skp2 are regulated by Akt, but the molecular mechanisms underlying these effects remain only partly understood. The scaffolding protein Ezrin-Binding Phosphoprotein of 50 kDa (EBP50) contains two PDZ domains and plays a critical role in the development of neointimal hyperplasia. Here we report that EBP50 directly binds Skp2 via its first PDZ domain. Moreover, EBP50 is phosphorylated by Akt on Thr-156 within the second PDZ domain, an event that allosterically promotes binding to Skp2. The interaction with EBP50 causes cytoplasmic localization of Skp2, increases Skp2 stability and promotes proliferation of primary vascular smooth muscle cells. Collectively, these studies define a novel regulatory mechanism contributing to aberrant cell growth and highlight the importance of scaffolding function of EBP50 in Akt-dependent cell proliferation.
Asunto(s)
Fosfoproteínas/metabolismo , Proteínas Proto-Oncogénicas c-akt/metabolismo , Proteínas Quinasas Asociadas a Fase-S/metabolismo , Intercambiadores de Sodio-Hidrógeno/metabolismo , Animales , Proliferación Celular , Células Cultivadas , Humanos , Ratones , Fosfoproteínas/química , Fosforilación , Unión Proteica , Estabilidad Proteica , Proteínas Proto-Oncogénicas c-akt/química , Proteínas Quinasas Asociadas a Fase-S/química , Intercambiadores de Sodio-Hidrógeno/químicaRESUMEN
AIMS: 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. METHODS: We examined expression of the toxin receptor (SV2) and its cleavage targets (SNAP-25 and SNAP-23) within urothelium as well as effects of the toxin on mechanically evoked release of ATP from cultured rat urothelial cells. ATP release was measured using the luciferin-luciferase assay; we examined expression of SNAP-23 and -25 in urothelial cells and mucosa of rat and human bladders. RESULTS: BoNT/A (1.5 U; 1-3 hr) blocked hypotonic evoked release of urothelial ATP, without affecting morphology. The expression of protein targets for BoNT/A binding (SV2) was detected in human and rat bladder mucosa and catalytic action (SNAP-23, -25) in urothelial cells and mucosa (differed in intensity) from rat and human bladder. Incubation of cultured (rat) urothelial cells with BoNT/A decreased expression levels of both SNAP-23 (44%) and SNAP-25 (80%). CONCLUSIONS: Our findings reveal that the bladder urothelium expresses the intracellular targets and the binding protein for cellular uptake of BoNT/A; and that the toxin is able to suppress the levels of these targets as well as hypotonic-evoked ATP release. These data raise the possibility that intravesical treatment with BoNT/A suppresses bladder reflex and sensory mechanisms by affecting a number of urothelial functions including release of transmitters.
Asunto(s)
Inhibidores de la Liberación de Acetilcolina/farmacología , Adenosina Trifosfato/metabolismo , Toxinas Botulínicas Tipo A/farmacología , Vejiga Urinaria/efectos de los fármacos , Urotelio/efectos de los fármacos , Inhibidores de la Liberación de Acetilcolina/uso terapéutico , Animales , Toxinas Botulínicas Tipo A/uso terapéutico , Células Cultivadas , Humanos , Glicoproteínas de Membrana/metabolismo , Contracción Muscular/efectos de los fármacos , Músculo Liso/efectos de los fármacos , Proteínas del Tejido Nervioso/metabolismo , Ratas , Proteína 25 Asociada a Sinaptosomas/metabolismo , Vejiga Urinaria/metabolismo , Vejiga Urinaria Hiperactiva/tratamiento farmacológico , Vejiga Urinaria Hiperactiva/metabolismo , Urotelio/metabolismo , Proteínas de Transporte Vesicular/metabolismoRESUMEN
The interaction between vascular cells and macrophages is critical during vascular remodeling. Here we report that the scaffolding protein, ezrin-binding phosphoprotein 50 (EBP50), is a central regulator of macrophage and vascular smooth muscle cells (VSMC) function. EBP50 is up-regulated in intimal VSMC following endoluminal injury and promotes neointima formation. However, the mechanisms underlying these effects are not fully understood. Because of the fundamental role that inflammation plays in vascular diseases, we hypothesized that EBP50 mediates macrophage activation and the response of vessels to inflammation. Indeed, EBP50 expression increased in primary macrophages and VSMC, and in the aorta of mice, upon treatment with LPS or TNFα. This increase was nuclear factor-κB (NF-κB)-dependent. Conversely, activation of NF-κB was impaired in EBP50-null VSMC and macrophages. We found that inflammatory stimuli promote the formation of an EBP50-PKCζ complex at the cell membrane that induces NF-κB signaling. Macrophage activation and vascular inflammation after acute LPS treatment were reduced in EBP50-null cells and mice as compared with WT. Furthermore, macrophage recruitment to vascular lesions was significantly reduced in EBP50 knock-out mice. Thus, EBP50 and NF-κB participate in a feed-forward loop leading to increased macrophage activation and enhanced response of vascular cells to inflammation.
Asunto(s)
Retroalimentación Fisiológica , FN-kappa B/metabolismo , Fosfoproteínas/metabolismo , Intercambiadores de Sodio-Hidrógeno/metabolismo , Vasculitis/metabolismo , Animales , Aorta/metabolismo , Células Endoteliales de la Vena Umbilical Humana/efectos de los fármacos , Células Endoteliales de la Vena Umbilical Humana/metabolismo , Células Endoteliales de la Vena Umbilical Humana/fisiología , Humanos , Lipopolisacáridos/farmacología , Macrófagos/metabolismo , Macrófagos/fisiología , Ratones , Ratones Endogámicos C57BL , Miocitos del Músculo Liso/efectos de los fármacos , Miocitos del Músculo Liso/metabolismo , Miocitos del Músculo Liso/fisiología , Fosfoproteínas/genética , Proteína Quinasa C/metabolismo , Intercambiadores de Sodio-Hidrógeno/genética , Factor de Necrosis Tumoral alfa/farmacología , Vasculitis/etiologíaRESUMEN
Glucagon-like peptide-1 (GLP-1) is a natural agonist for GLP-1R, a G protein-coupled receptor (GPCR) on the surface of pancreatic ß 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 ß-amino acid residues. This backbone-modification approach may be useful for developing stabilized analogues of other peptide hormones.
Asunto(s)
Péptido 1 Similar al Glucagón/análogos & derivados , Péptido 1 Similar al Glucagón/farmacología , Receptores de Glucagón/agonistas , Secuencia de Aminoácidos , Animales , Células Cultivadas , Diabetes Mellitus Tipo 2/tratamiento farmacológico , Péptido 1 Similar al Glucagón/química , Péptido 1 Similar al Glucagón/metabolismo , Receptor del Péptido 1 Similar al Glucagón , Humanos , Ratones , Datos de Secuencia Molecular , Estabilidad ProteicaRESUMEN
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.
Asunto(s)
Hepatocitos/metabolismo , Óxido Nítrico Sintasa de Tipo II/metabolismo , Peroxisomas/metabolismo , Fosfoproteínas/metabolismo , Receptores Citoplasmáticos y Nucleares/metabolismo , Intercambiadores de Sodio-Hidrógeno/metabolismo , Animales , Hepatocitos/química , Hepatocitos/enzimología , Hígado/química , Hígado/enzimología , Hígado/metabolismo , Ratones , Ratones Noqueados , Microscopía Confocal , Microscopía Fluorescente , Receptor de la Señal 2 de Direccionamiento al Peroxisoma , Fosfoproteínas/genética , ARN Interferente Pequeño/genética , Receptores Citoplasmáticos y Nucleares/genética , Transducción de Señal , Intercambiadores de Sodio-Hidrógeno/genéticaRESUMEN
OBJECTIVE: The Ezrin-radixin-moesin-binding phosphoprotein 50 (EBP50) is a scaffolding protein known to regulate ion homeostasis in the kidney and intestine. Previous work showed that EBP50 expression increases after balloon injury in rat carotids. This study was designed to determine the role of EBP50 on vascular smooth muscle cells (VSMC) proliferation and the development of neointimal hyperplasia. METHODS AND RESULTS: Wire injury was performed in wild type (WT) and EBP50 knockout (KO) mice. Two weeks after injury, neointima formation was 80% lower in KO than in WT mice. Proliferation of KO VSMC was significantly lower than WT cells and overexpression of EBP50 increased VSMC proliferation. Akt activity and expression of S-phase kinase protein2 decreased in KO cells resulting in the stabilization of the cyclin-dependent kinase inhibitor, p21(cip1). Consequently, KO cells were arrested in G(0)/G(1) phase. Consistent with these observations, p21(cip1) was detected in injured femoral arteries of KO but not WT mice. No differences in apoptosis between WT and KO were observed. CONCLUSIONS: EBP50 is critical for neointima formation and induces VSMC proliferation by decreasing S-phase kinase protein2 stability, thereby accelerating the degradation of the cell cycle inhibitor p21(cip1).
Asunto(s)
Inhibidor p21 de las Quinasas Dependientes de la Ciclina/fisiología , Miocitos del Músculo Liso/citología , Miocitos del Músculo Liso/fisiología , Neointima/etiología , Fosfoproteínas/fisiología , Proteínas Quinasas Asociadas a Fase-S/fisiología , Intercambiadores de Sodio-Hidrógeno/fisiología , Animales , Proliferación Celular , Arteria Femoral/lesiones , Arteria Femoral/patología , Arteria Femoral/fisiopatología , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Neointima/patología , Neointima/fisiopatología , Fosfoproteínas/deficiencia , Fosfoproteínas/genética , Proteínas Proto-Oncogénicas c-akt/metabolismo , Ratas , Intercambiadores de Sodio-Hidrógeno/genéticaRESUMEN
The ezrin-radixin-moesin-binding phosphoprotein 50 (EBP50) is a PDZ-containing scaffolding protein that regulates a variety of physiological functions. In the vasculature, EBP50 promotes neointima formation following arterial injury. In this study the role of EBP50 on vascular smooth muscle cell (VSMC) migration was characterized. The spreading and motility of primary VSMC isolated from EBP50 knockout (KO) mice were significantly reduced compared to wild-type (WT) cells. EBP50-null VSMC had fewer and larger focal adhesions than wild-type cells. Assembly and disassembly of focal adhesion-assessed by live-cell total internal reflection fluorescence imaging-in response to epidermal growth factor (EGF) were significantly reduced in KO cells. Immunoprecipitation experiments showed that EBP50 interacts with EGF receptor via the PDZ2 domain and with focal adhesion kinase (FAK) via the C-terminal ERM domain. EBP50 promoted the formation of a complex containing both EGF receptor and FAK. Phosphorylation of Tyr-925 of FAK in response to EGF was significantly reduced in KO cell compared to WT cells. The residence time of FAK in focal adhesions-determined by fluorescence recovery after photobleaching-was increased in WT cells. Collectively, these studies indicate that EBP50, by scaffolding EGF receptor and FAK, facilitates activation of FAK, focal adhesion turnover, and migration of VSMC.
Asunto(s)
Vasos Sanguíneos/metabolismo , Movimiento Celular , Adhesiones Focales/metabolismo , Miocitos del Músculo Liso/metabolismo , Fosfoproteínas/metabolismo , Intercambiadores de Sodio-Hidrógeno/metabolismo , Animales , Línea Celular , Movimiento Celular/efectos de los fármacos , Movimiento Celular/genética , Factor de Crecimiento Epidérmico/farmacología , Receptores ErbB/metabolismo , Quinasa 1 de Adhesión Focal/metabolismo , Adhesiones Focales/efectos de los fármacos , Adhesiones Focales/genética , Ratones , Ratones Noqueados , Miocitos del Músculo Liso/efectos de los fármacos , Fosfoproteínas/genética , Fosforilación/efectos de los fármacos , Unión Proteica , Intercambiadores de Sodio-Hidrógeno/genéticaRESUMEN
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.
Asunto(s)
Proteínas Portadoras/metabolismo , Mitógenos/metabolismo , Músculo Liso Vascular/metabolismo , Miocitos del Músculo Liso/citología , Fosfoproteínas/metabolismo , Receptor de Hormona Paratiroídea Tipo 1/metabolismo , Angioplastia , Animales , Arterias Carótidas/metabolismo , Arterias Carótidas/cirugía , Proliferación Celular , Endocitosis , Células HEK293 , Humanos , Masculino , Modelos Biológicos , Neointima/metabolismo , Neointima/patología , Transporte de Proteínas , Ratas , Ratas Sprague-Dawley , Transducción de Señal , Intercambiadores de Sodio-Hidrógeno , Regulación hacia ArribaRESUMEN
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.
Asunto(s)
Adenosina Trifosfato/metabolismo , Membrana Celular/metabolismo , Receptores Purinérgicos P2/metabolismo , Vejiga Urinaria/citología , Urotelio , Adenosina Trifosfato/agonistas , Animales , Apirasa/metabolismo , Calcio/metabolismo , Proteínas Quinasas Dependientes de AMP Cíclico/metabolismo , Capacidad Eléctrica , Endocitosis/fisiología , Exocitosis/fisiología , Femenino , Técnicas In Vitro , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Agonistas del Receptor Purinérgico P2 , Fosfato de Piridoxal/análogos & derivados , Fosfato de Piridoxal/metabolismo , Conejos , Receptores Purinérgicos P2/genética , Receptores Purinérgicos P2X2 , Receptores Purinérgicos P2X3 , Transducción de Señal/fisiología , Urotelio/metabolismo , Urotelio/ultraestructuraRESUMEN
The control and regulation of the lower urinary tract are partly mediated by purinergic signaling. This study investigated the distribution and function of P2Y receptors in the rat urinary bladder. Application of P2Y agonists to rat urothelial cells evoked increases in intracellular calcium; the rank order of agonist potency (pEC(50) +/- SE) was ATP (5.10 +/- 0.07) > UTP (4.91 +/- 0.14) > UTPgammaS (4.61 +/- 0.16) = ATPgammaS (4.70 +/- 0.05) > 2-methylthio adenosine 5'-diphosphate = 5'-(N-ethylcarboxamido)adenosine = ADP (<3.5). The rank order potency for these agonists indicates that urothelial cells functionally express P2Y(2)/P2Y(4) receptors, with a relative lack of contribution from other P2Y or adenosine receptors. Real-time PCR, Western blotting, and immunocytochemistry confirmed the expression of P2Y(2) and to a lesser extent P2Y(4) in the urothelium. Immunocytochemical studies revealed expression of P2Y(2) staining in all layers of the urothelium, with relative absence of P2Y(4). P2Y(2) staining was also present in suburothelial nerve bundles and underlying detrusor smooth muscle. Addition of UTP and UTPgammaS was found to evoke ATP release from cultured rat urothelial cells. These findings indicate that cultured rat urothelial cells functionally express P2Y(2)/P2Y(4) receptors. Activation of these receptors could have a role in autocrine and paracrine signaling throughout the urothelium. This could lead to the release of bioactive mediators such as additional ATP, nitric oxide, and acetylcholine, which can modulate the micturition reflex by acting on suburothelial myofibroblasts and/or pelvic afferent fibers.
Asunto(s)
Receptores Purinérgicos P2/genética , Vejiga Urinaria/fisiología , Urotelio/fisiología , Adenosina Trifosfato/farmacología , Animales , Calcio/fisiología , Células Cultivadas , Cartilla de ADN , Regulación de la Expresión Génica , Inmunohistoquímica , Reacción en Cadena de la Polimerasa , Ratas , Ratas Sprague-Dawley , Receptores Purinérgicos P2Y2 , Vejiga Urinaria/citología , Vejiga Urinaria/efectos de los fármacos , Urotelio/citología , Urotelio/efectos de los fármacosRESUMEN
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 4alpha-phorbol 12,13-didecanoate (4alpha-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 4alpha-PDD (10-100 microM) 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 microM), 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 micromol/kg), or urethane anesthesia, but was unaffected by capsaicin pretreatment (100 mg/kg s.c.) or denervation of the urethral sphincter. 4Alpha-PDD (1-100 microM) did not alter the contractility to electrical stimulation of excised bladder strips. We conclude that activation of urothelial TRPV4 by 4alpha-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.
Asunto(s)
Contracción Muscular/efectos de los fármacos , Ésteres del Forbol/farmacología , Canales Catiónicos TRPV/biosíntesis , Vejiga Urinaria/efectos de los fármacos , Micción/efectos de los fármacos , Urotelio/efectos de los fármacos , Animales , Femenino , Immunoblotting , Inmunohistoquímica , Técnicas In Vitro , Masculino , Músculo Liso/efectos de los fármacos , Músculo Liso/fisiología , Ratas , Ratas Sprague-Dawley , Reflejo/efectos de los fármacos , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Canales Catiónicos TRPV/agonistas , Vejiga Urinaria/fisiología , Urotelio/metabolismoRESUMEN
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.