Susan Meyers

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.

EFFECTS OF VITAMIN D (CALCITRIOL) ON TRANSITIONAL CELL CARCINOMA OF THE BLADDER IN VITRO AND IN VIVO

PURPOSE Vitamin D (calcitriol) has significant antiproliferative effects on various tumor cells in vitro and in vivo. In the clinical situation a major impediment to systemic administration of calcitriol is the side effect of hypercalcemia. To test the potential usefulness of calcitriol for bladder cancer treatment, we studied the antiproliferative effect of vitamin D on 2 human bladder cancer cell lines, 253j and T-24, in vitro. We also examined the in vivo effects of calcitriol in an animal model of bladder cancer using intravesical administration to avoid the toxicity of systemic calcitriol therapy. MATERIALS AND METHODS The presence of vitamin D receptors in normal and neoplastic human bladder tissue, and tumor cells T-24 and 253j was determined by immunoblot analysis. Tumor cell proliferation in the presence or absence of calcitriol was determined using a crystal violet assay. Calcitriol induced apoptosis was determined by morphology, polyadenosine diphosphate ribose polymerase cleavage and annexin V binding. In vivo studies were performed by weekly intravesical instillation of calcitriol in female Fischer 344 rats after induction of tumors by N-methyl nitrosourea. Calcitriol administration was started 3 weeks after tumor induction for 7 doses at weekly intervals. RESULTS Normal and neoplastic human bladder tissue, and the cell lines expressed vitamin D receptors. In the 253j and T-24 cell lines proliferation was significantly inhibited by calcitriol. Progressive cleavage of full length polyadenosine diphosphate ribose polymerase was observed in calcitriol treated cells starting as early as 4 hours after exposure. Similar changes were not observed in the control cells treated with vehicle (ethanol) alone. After 24 hours of treatment with calcitriol 45.8% of 253j cells bound annexin compared to 16.5% of control cells (chi-square p <0.001). Of the control animals 66% developed bladder tumors and 55% of the animals treated with calcitriol early (3 weeks) after tumor induction developed bladder tumors. Almost all of the tumors that developed in the calcitriol group were unifocal, and only 20% were invasive compared to 50% of those in the control animals. CONCLUSIONS These results demonstrate that calcitriol inhibits proliferation and induces apoptosis in human bladder tumor cells in vitro, and may have therapeutic potential in bladder cancer. In vivo studies using an N-methylnitrosourea induced model of bladder cancer demonstrate that early institution of intravesical calcitriol therapy after carcinogen exposure results in fewer tumors, which are also less likely to be multifocal, high grade or invasive. With our protocol a short course of intravesical calcitriol administration did not result in any significant toxicity.

Enhancing early bladder cancer detection with fluorescence-guided endoscopic optical coherence tomography.

We report an experimental study of the possibility of enhancing early bladder cancer diagnosis with fluorescence-image-guided endoscopic optical coherence tomography (OCT). After the intravesical instillation of a 10% solution of 5-aminolevulinic acid, simultaneous fluorescence imaging (excitation of 380-420 nm, emission of 620-700 nm) and OCT are performed on rat bladders to identify the photochemical and morphological changes associated with uroepithelial tumorigenesis. The preliminary results of our ex vivo study reveal that both fluorescence and OCT can identify early uroepithelial cancers, and OCT can detect precancerous lesions (e.g., hyperplasia) that fluorescence may miss. This suggests that a cystoscope combining 5-aminolevulinic acid fluorescence and OCT imaging has the potential to enhance the efficiency and sensitivity of early bladder cancer diagnosis.

Detection of tumorigenesis in rat bladders with optical coherence tomography

Optical coherence tomography (OCT) is a novel technique that enables noninvasive cross-sectional imaging of biological tissues. Because of its high resolution (approximately 10 microm), superior dynamic range (140 dB in our case) and up to 2-3 mm penetration depth, OCT is potentially useful for noninvasive screening of superficial lesions. Bladder cancer arises within the transitional epithelium. Despite the ability to visualize the epithelium via cystoscopy, it is often difficult to detect early epithelial cancers and to determine their penetration to the underlying layers. To investigate the potential of OCT to enhance imaging of bladder cancers and other epithelial lesions, we applied OCT to normal and diseased bladder epithelium, and correlated the results with histological findings. OCT images of porcine bladder (a close homolog of human bladder) confirm the ability of this method to image human tissues. To determine whether OCT can track the course of bladder cancer, a standard rat model of bladder cancer in which Fisher rats are exposed to methyl-nitroso-urea (MNU), was followed both with OCT and histological studies. Our results show that the micro morphology of porcine bladder such as the urothelium, submucosa and muscles is identified by OCT and well correlated with the histological evaluations. OCT detected edema, inflammatory infiltrates, and submucosal blood congestion as well as the abnormal growth of urothelium (e.g., papillary hyperplasia and carcinomas). By contrast, surface imaging, which resembles cystoscopy, provided far less sensitivity and resolution than OCT. This is the first OCT study of any tumor documented in a systematic fashion, and the results suggest the potential of OCT for the noninvasive diagnosis of both bladder inflammatory lesions and early urothelial abnormalities, which conventional cystoscopy often misses, by imaging characterization of the increases in urothelial thickening and backscattering. However, because of the depth limitation, OCT may have limited applications in staging the invasion of higher-state urothelial cancers, especially for papillary carcinomas.

Disruption of guinea pig urinary bladder permeability barrier in noninfectious cystitis

Although most cell membranes permit rapid flux of water, small nonelectrolytes, and ammonia, the apical membranes of bladder epithelial umbrella cells, which form the bladder permeability barrier, exhibit strikingly low permeabilities to these substances. In cystitis, disruption of the bladder permeability barrier may irritate the bladder wall layers underlying the epithelium, causing or exacerbating inflammation, and increasing urinary frequency, urgency, and bladder pain. To determine the effects of inflammation on the integrity of the permeability barrier, guinea pigs were sensitized with ovalbumin, and the bladders were exposed subsequently to antigen by instillation on the urinary side. Inflammation of the bladder wall markedly reduced transepithelial resistance of dissected epithelium mounted in Ussing chambers and increased water and urea permeabilities modestly at 2 h and more strikingly at 24 h after induction of the inflammation. Transmission and scanning electron microscopy of bladders at 30 min and 24 h after antigen exposure revealed disruption of tight junctions, denuding of patches of epithelium, and occasional loss of apical membrane architecture. These permeability and structural effects did not occur in nonsensitized animals in which the bladders were exposed to antigen and in sensitized animals exposed to saline vehicle rather than antigen. These results demonstrate that inflammation of the underlying muscle and lamina propria can disrupt the bladder permeability barrier by damaging tight junctions and apical membranes and causing sloughing of epithelial cells. Leakage of urinary constituents through the damaged epithelium may then exacerbate the inflammation in the underlying muscle layers.Although most cell membranes permit rapid flux of water, small nonelectrolytes, and ammonia, the apical membranes of bladder epithelial umbrella cells, which form the bladder permeability barrier, exhibit strikingly low permeabilities to these substances. In cystitis, disruption of the bladder permeability barrier may irritate the bladder wall layers underlying the epithelium, causing or exacerbating inflammation, and increasing urinary frequency, urgency, and bladder pain. To determine the effects of inflammation on the integrity of the permeability barrier, guinea pigs were sensitized with ovalbumin, and the bladders were exposed subsequently to antigen by instillation on the urinary side. Inflammation of the bladder wall markedly reduced transepithelial resistance of dissected epithelium mounted in Ussing chambers and increased water and urea permeabilities modestly at 2 h and more strikingly at 24 h after induction of the inflammation. Transmission and scanning electron microscopy of bladders at 30 min and 24 h after antigen exposure revealed disruption of tight junctions, denuding of patches of epithelium, and occasional loss of apical membrane architecture. These permeability and structural effects did not occur in nonsensitized animals in which the bladders were exposed to antigen and in sensitized animals exposed to saline vehicle rather than antigen. These results demonstrate that inflammation of the underlying muscle and lamina propria can disrupt the bladder permeability barrier by damaging tight junctions and apical membranes and causing sloughing of epithelial cells. Leakage of urinary constituents through the damaged epithelium may then exacerbate the inflammation in the underlying muscle layers.

Urinary Bladder Function and Somatic Sensitivity in Vasoactive Intestinal Polypeptide (VIP) −/− Mice

Vasoactive intestinal polypeptide (VIP) is an immunomodulatory neuropeptide widely distributed in neural pathways that regulate micturition. VIP is also an endogenous anti-inflammatory agent that has been suggested for the development of therapies for inflammatory disorders. In the present study, we examined urinary bladder function and hindpaw and pelvic sensitivity in VIP−/− and littermate wildtype (WT) controls. We demonstrated increased bladder mass and fewer but larger urine spots on filter paper in VIP−/− mice. Using cystometry in conscious, unrestrained mice, VIP−/− mice exhibited increased void volumes and shorter intercontraction intervals with continuous intravesical infusion of saline. No differences in transepithelial resistance or water permeability were demonstrated between VIP−/− and WT mice; however, an increase in urea permeability was demonstrated in VIP−/− mice. With the induction of bladder inflammation by acute administration of cyclophosphamide, an exaggerated or prolonged bladder hyperreflexia and hindpaw and pelvic sensitivity were demonstrated in VIP−/− mice. The changes in bladder hyperreflexia and somatic sensitivity in VIP−/− mice may reflect increased expression of neurotrophins and/or proinflammatory cytokines in the urinary bladder. Thus, these changes may further regulate the neural control of micturition.

Protamine sulfate-induced cystitis: a model of selective cytodestruction of the urothelium.

monary fibrosis. Because mast cell enzymes have been implicated in the fibrotic response, we sought to examine the possible regulatory role of mast cells in bladder wall fibrosis. Hence, the objectives of this study were to determine if there was an increase in inflammatory mast cell number and location in bladder wall tissue from myelodysplasia patients, and to determine if the mast cell proteases, chymase and tryptase, could alter connective tissue expression in bladder wall cells. Transmural tissue sections from human control (n 5 5) and myelodysplastic (n 5 7) bladders were stained with mast cell–specific serine protease antibodies (antitryptase, antichymase) to determine the number, location, and state of degranulation of mast cells. Human bladder lamina propria fibroblasts and detrusor smooth muscle cells were isolated from control tissue collagenase digestion, treated in vitro with recombinant chymase and tryptase, and analyzed by enzyme-linked immunosorbent assay for alterations in extracellular matrix proteins (collagens I and III, and elastin). Bladders from myelomeningocele patients were characterized by increased numbers of mast cells within detrusor muscle bundles infiltrated with connective tissue. Regions of detrusor muscle adjacent to the lamina propria showed evidence of mast cell degranulation and increased connective tissue deposition. Tryptase treatment resulted in a decrease in both type III collagen and elastin in both fibroblasts and smooth muscle cells. In contrast, chymase treatment resulted in an increase in both type III and elastin. Expression of type I collagen was variable depending on the cell type treated and the specific enzyme. Denaturation of the enzymes by heat treatment resulted in a return to control levels. These studies show that bladder fibrosis may be mediated, in part, by mast cell products altering the connective tissue milieu of the bladder wall.

Uroplakins Do Not Restrict CO2 Transport through Urothelium

Background: The tightness of various membrane barriers to CO2 is of unknown molecular origin. Results: The bladder tissue lacks carbonic anhydrase. The resulting low intra-epithelial CO2 concentration gives rise to the apparent CO2 impermeability. Conclusion: Uroplakins do not act to decrease transepithelial CO2 flux. Significance: Enzymatic regulation of CO2 abundance rules out that aquaporins significantly contribute to the maintenance of acid base homeostasis. Lipid bilayers and biological membranes are freely permeable to CO2, and yet partial CO2 pressure in the urine is 3–4-fold higher than in blood. We hypothesized that the responsible permeability barrier to CO2 resides in the umbrella cell apical membrane of the bladder with its dense array of uroplakin complexes. We found that disrupting the uroplakin layer of the urothelium resulted in water and urea permeabilities (P) that were 7- to 8-fold higher than in wild type mice with intact urothelium. However, these interventions had no impact on bladder PCO2 (∼1.6 × 10−4 cm/s). To test whether the observed permeability barrier to CO2 was due to an unstirred layer effect or due to kinetics of CO2 hydration, we first measured the carbonic anhydrase (CA) activity of the bladder epithelium. Finding none, we reduced the experimental system to an epithelial monolayer, Madin-Darby canine kidney cells. With CA present inside and outside the cells, we showed that PCO2 was unstirred layer limited (∼7 × 10−3 cm/s). However, in the total absence of CA activity PCO2 decreased 14-fold (∼ 5.1 × 10−4 cm/s), indicating that now CO2 transport is limited by the kinetics of CO2 hydration. Expression of aquaporin-1 did not alter PCO2 (and thus the limiting transport step), which confirmed the conclusion that in the urinary bladder, low PCO2 is due to the lack of CA. The observed dependence of PCO2 on CA activity suggests that the tightness of biological membranes to CO2 may uniquely be regulated via CA expression.

Studies on localization and function of annexin A4a within urinary bladder epithelium using a mouse knockout model

Annexin A4 (anxA4) is a member of the Ca(2+)-dependent membrane-binding family of proteins implicated in the regulation of ion conductances, Ca(2+) homeostasis, and membrane trafficking. We demonstrate, in mice, that annexins 1-6 are present in whole bladder and exhibit differential expression in the urothelium. An anxA4a-knockout (anxA4a(-/-)) mouse model shows no protein in the urothelium by immunofluorescence and immunoblotting. In wild-type bladders, anxA4a in umbrella cells showed uniform cytoplasmic staining and some association with the nuclear membrane. Application of a hydrostatic pressure to bladders mounted in Ussing chambers resulted in redistribution of anxA4a from cytoplasm to cellular boundaries in the basal and intermediate cells but not in superficial umbrella cells. We hypothesized that anxA4a might be important for barrier function or for stretch-activated membrane trafficking. To test these hypotheses, we conducted a series of functional and morphological analyses on bladders from control and anxA4a(-/-) animals. The transepithelial resistances, water permeabilities, and urea permeabilities of anxA4a(-/-) bladders were not different from controls, indicating that barrier function was intact. Membrane trafficking in response to hydrostatic pressure as measured by capacitance increases was also normal for anxA4a(-/-) bladders. Cystometrograms performed on live animals showed that voiding frequency and intrabladder pressures were also not different. There were no differences in bladder surface morphology or cellular architecture examined by scanning and transmission electron microscopy, respectively. We conclude that loss of anxA4 from the urothelium does not affect barrier function, membrane trafficking, or normal bladder-voiding behavior.

Diminished Sympathetic Responsiveness in Nephrectomized Rats-Role of the Renin Angiotensin System

Experiments were conducted in pithed Wistar rats to assess the effects of nephrectomy on the responsiveness of the cardiovascular system to sympathetic neurohumoral stimuli. Blood pressure and heart rate increases produced in response to stimulation of the spinal sympathetic outflow and to norepinephrine were compared in nephrectomized (NXR) and sham operated animals (SOR). Both the blood pressure (BP) and heart rate (HR) increases to nerve stimulation were markedly attenuated in the NXR, however, only the reduction in BP responses could be attributed to the absence of a functional renin angiotensin system. Infusion of angiotensin II (10 ng/kg/min) in NXR enhanced the neurally mediated increments in BP to the extent that the responses were not different from SOR. Angiotensin II administration also enhanced BP responses in SOR but to a lesser extent than in NXR. HR responses were not altered by angiotensin in NXR or SOR. Blockade of the renin angiotensin system in SOR with the converting enzyme inhibitor, captopril, reduced BP responses to the same level as NXR. In contrast, HR increments were not altered by captopril. BP but not HR increments to norepinephrine were significantly reduced in NXR. Infusion of angiotensin II restored the BP responses to a level equal to SOR; HR responses were not affected. In addition, captopril reduced the norepinephrine responses of SOR but not NXR. Thus the results of the present study indicate that endogenously formed angiotensin facilitates sympathetically mediated vasoconstrictor activity but does not influence heart rate responses. Therefore, the attenuation of neurally elicited increases in HR observed in NXR does not appear to be acutely related to reduced levels of angiotensin II.