Mouse Urinary Bladder Research Articles

Effect of Recombinant Human Keratinocyte Growth Factor (rHuKGF, Palifermin) on Radiation-Induced Mouse Urinary Bladder Dysfunction

Purpose/Objective: Radiation-induced changes in urinary bladder storage capacity are a frequent side effect of pelvic radiotherapy. In mice, early functional changes in bladder occur within the first month after irradiation in two phases, with peaks around day 7 (acute phase 1) and day 23 (acute phase 2). Acute changes are followed by a dose-dependent latent time period and by a subsequent and irreversible late response phase. Recently, administration of growth factors, such as keratinocyte growth factor (KGF), has been shown to modify radiation effects. Therefore, the present study was initiated to identify the effect of Palifermin (rHuKGF) on acute and late radiation effects in the mouse urinary bladder Materials/Methods: For this, graded radiation doses were applied to generate full dose-effect curves. Changes in bladder function, measured by transurethral cystometry, were assessed. Conclusions: In conclusion, early steps of the radiation-induced acute changes in the mouse urinary bladder can be modified with a single administration of Palifermin (rHuKGF) before irradiation. However, the optimum dosage and also the influence of the repeated injections have to be identified in further experiments. Moreover, studies into KGF-effects during fractionated irradiation must be performed.

Two-step Ca2+ intracellular release underlies excitation-contraction coupling in mouse urinary bladder myocytes

The relative contributions of Ca(2+)-induced Ca(2+) release (CICR) versus Ca(2+) influx through voltage-dependent Ca(2+) channels (VDCCs) to excitation-contraction coupling has not been defined in most smooth muscle cells (SMCs). The present study was undertaken to address this issue in mouse urinary bladder (UB) smooth muscle cells (UBSMCs). Confocal Ca(2+) images were obtained under voltage- or current-clamp conditions. When UBSMCs were activated by a 30-ms depolarization to 0 mV, intracellular Ca(2+) concentration ([Ca(2+)](i)) increased in several small, discrete areas just beneath the cell membrane. These Ca(2+) "hot spots" then spread slowly through the myoplasm as Ca(2+) waves, which continued even after repolarization. Shorter depolarizations (5 ms) elicited only a few Ca(2+) sparks, which declined quickly. The number of Ca(2+) sparks, or hot spots, was closely related to the depolarization duration in the range of approximately 5-20 ms. There was an apparent threshold depolarization duration of approximately 10 ms within which to induce enough Ca(2+) transients to spread globally and then induce a contraction. Application of 100 microM ryanodine to the pipette solution did not change the resting [Ca(2+)](i) or the VDCC current, but it did abolish Ca(2+) hot spots elicited by depolarization. Application of 3 microM xestospongin C reduced ACh-induced Ca(2+) release but did not affect depolarization-induced Ca(2+) events. The addition of 100 microM ryanodine to tissue segments markedly reduced the amplitude of contractions triggered by direct electrical stimulation. In conclusion, global [Ca(2+)](i) rise triggered by a single action potential is not due mainly to Ca(2+) influx through VDCCs but is attributable to the subsequent two-step CICR.

Early and long-term effects of radiation on intercellular adhesion molecule 1 (ICAM-1) expression in mouse urinary bladder endothelium

The aim was to assess the effect of irradiation on intercellular adhesion molecule 1 (ICAM-1) expression in endothelial cells of vessels in mouse urinary bladder and to compare endothelial ICAM-1 expression with changes in bladder function (storage capacity) during the early and late radiation response phases. Female C3H/Neu mice were irradiated with doses of either 20 or 0 Gy. For assessment of ICAM-1 expression, which was measured by the intensity of the immunohistochemical staining signal in bladder endothelium, an arbitrary semiquantitative score (0 – 3) was applied. Bladder storage function was assessed by transurethral cystotonometry. A positive functional radiation response, defined as a reduction in bladder capacity by > 50%, between days 0 and 15 or 16 and 30 was found in 40 and 64% of the animals, respectively. A late functional response was observed in 71% of the animals sacrificed after day 180. Minor constitutive expression of ICAM-1 was observed in bladder endothelial cells. After irradiation, an increase in staining signal by day 2, with a maximum on day 4, and on days 16 – 28 was found, which preceded the functional radiation effects. A permanent increase in ICAM-1 staining signal was observed in the late phase on top of an age-related rise. ICAM-1 expression was significantly higher in animals with a positive late response on day 90, i.e. during the initial late phase. Irradiation induces significant early and chronic variations in ICAM-1 expression in bladder endothelium, which preceded the functional response. This suggests that endothelial ICAM-1 is involved in the pathogenesis of both the early and late phases of radiation-induced urinary bladder effects.

Negative feedback regulation of nerve-mediated contractions by KCachannels in mouse urinary bladder smooth muscle

When the urinary bladder is full, activation of parasympathetic nerves causes release of neurotransmitters that induce forceful contraction of the detrusor muscle, leading to urine voiding. The roles of ion channels that regulate contractility of urinary bladder smooth muscle (UBSM) in response to activation of parasympathetic nerves are not well known. The present study was designed to characterize the role of large (BK)- and small-conductance (SK) Ca(2+)-activated K(+) (K(Ca)) channels in regulating UBSM contractility in response to physiological levels of nerve stimulation in UBSM strips from mice. Nerve-evoked contractions were induced by electric field stimulation (0.5-50 Hz) in isolated strips of UBSM. BK and SK channel inhibition substantially increased the amplitude of nerve-evoked contractions up to 2.45 +/- 0.12- and 2.99 +/- 0.25-fold, respectively. When both SK and BK channels were inhibited, the combined response was additive. Inhibition of L-type voltage-dependent Ca(2+) channels (VDCCs) in UBSM inhibited nerve-evoked contractions by 92.3 +/- 2.0%. These results suggest that SK and BK channels are part of two distinct negative feedback pathways that limit UBSM contractility in response to nerve stimulation by modulating the activity of VDCCs. Dysfunctional regulation of UBSM contractility by alterations in BK/SK channel expression or function may underlie pathologies such as overactive bladder.

The M2 Muscarinic Receptor Mediates Contraction through Indirect Mechanisms in Mouse Urinary Bladder

We investigated the contractile role of M2 muscarinic receptors in mouse urinary bladder. When measured in the absence of other agents, contractions elicited to the muscarinic agonist oxotremorine-M exhibited properties consistent with that expected for an M3 response in urinary bladder from wild-type and M2 knockout (KO) mice. Evidence for a minor M2 receptor-mediated contraction was revealed by a comparison of responses in M3 knockout and M2/M3 double knockout mice. Treatment of wild-type and M2 knockout urinary bladder with N-2-chloroethyl-4-piperidinyl diphenylacetate (4-DAMP mustard) caused a large inhibition of the muscarinic contractile response. The residual contractions were much smaller in M2 knockout bladder compared with wild type, suggesting that M2 receptors rescue the muscarinic contractile response in wild-type bladder following inactivation of M3 receptors with 4-DAMP mustard. When measured in the presence of prostaglandin F2alpha and isoproterenol or forskolin, oxotremorine-M mediated a potent contractile response in urinary bladder from M3 KO mice. This response exhibited an M2 profile in competitive antagonism studies and was completely absent in M2/M3 KO mice. Following 4-DAMP mustard treatment, oxotremorine-M elicited a contractile response in wild-type urinary bladder in the presence of KCl and isoproterenol or forskolin, and this response was diminished in M2 KO mice. Our results show that the M2 receptor mediates contractions indirectly in the urinary bladder by enhancing M3 receptor-mediated contractions and inhibiting relaxation. We also show that it is difficult to detect M2 receptor function in competitive antagonism studies under conditions where a simultaneous activation of M2 and M3 receptors occurs.

476 oral Radiation-induced changes in cyclooxygenase-2 expression in mouse urinary bladder

476 oral Radiation-induced changes in cyclooxygenase-2 expression in mouse urinary bladder

Oxidant driven signaling pathways during diabetes: role of Rac1 and modulation of protein kinase activity in mouse urinary bladder

Background. – Urinary bladder dysfunction is a complication in diabetes but the mechanisms involved are undefined. Here, we investigated roles of oxidative stress and oxidant driven signaling pathways in a murine model of diabetes, with an emphasis on urothelial vs. smooth muscle regional changes. Methods. – Mice were dosed with streptozotocin (150 mg/kg) or vehicle and studied at 5 weeks. Functional changes were assessed by in vitro cystometry. Immunohistochemical methods and automated digital imaging was used for morphometric and histochemical analysis of bladder tissue regions. Results. – We detected significant increases in protein 3-nitrotyrosine in both urothelium and smooth muscle regions during diabetes, demonstrating an increased prevalence of reactive nitrogen species. In light of nitric oxide synthase (NOS) isoforms as potential contributors to increased protein nitration, all three NOS isoforms were studied; region specific increases in NOS1 (urothelium and smooth muscle), NOS2 (urothelium only) but no alterations in NOS3 isoform were detected during diabetes. In contrast, p21-Rac1 (coordinating protein of NADPH oxidase) was significantly increased only in smooth muscle (diabetic vs. controls). We also investigated phosphorylation of ERK, JNK, p38 and Akt using immunohistochemical techniques; each of these was increased during diabetes but with different distributions in the two major regions of bladder tissues viz the smooth muscle and urothelium. Conclusions. – The STZ mouse model of diabetes exhibits bladder dysfunction and structural changes similar to human. Reactive nitrogen species formation occurs in this setting and region specific assessments also revealed that urothelial changes and smooth muscle changes are discrete with respect to mechanisms of reactive nitrogen species (increased production of NO vs. superoxide anion) and activation of oxidant related stress signaling pathways.

Muscarinic receptors mediating contraction of female mouse urinary bladder: effects of oestrogen

Muscarinic receptors mediating contraction of bladder detrusor muscle from female mice were examined. Mice were untreated (A) or treated with oestradiol cypionate (200 μg/kg) 24 h (B) or 96 h (C) before experimentation, or were pregnant (day 17) (D). Saturation radioligand binding experiments using [ 3H]quinuclidinyl benzilate ([ 3H] QNB) indicated similar muscarinic receptor densities and affinities in bladders from groups A and B. Neither oestrogen treatment nor pregnancy altered p D 2 estimates for methacholine. Maximum responses to methacholine and high-K + physiological salt solution (KPSS) were significantly greater ( P<0.05) in tissues from groups C and D than in A and B. Potencies of other muscarinic receptor agonists were similar in groups A and B with an order of acetylcholine plus physostigmine (10 μM)≈methacholine plus physostigmine (10 μM)>methacholine≈acetylcholine>bethanechol. Antagonist p K B estimates were similar in bladders from groups A and B with a rank order of: atropine≥4-diphenyl acetoxy- N-methyl piperidine methiodide>parafluorohexahydrosiladifenidol≈pirenzepine>himbacine, implicating muscarinic M 1 and/or M 5 as well as muscarinic M 3 receptors in mediating methacholine-induced bladder contraction.

Properties of a tonically active, sodium-permeable current in mouse urinary bladder smooth muscle.

Urinary bladder smooth muscle (UBSM) elicits depolarizing action potentials, which underlie contractile events of the urinary bladder. The resting membrane potential of UBSM is approximately -40 mV and is critical for action potential generation, with hyperpolarization reducing action potential frequency. We hypothesized that a tonic, depolarizing conductance was present in UBSM, functioning to maintain the membrane potential significantly positive to the equilibrium potential for K(+) (E(K); -85 mV) and thereby facilitate action potentials. Under conditions eliminating the contribution of K(+) and voltage-dependent Ca(2+) channels, and with a clear separation of cation- and Cl(-)-selective conductances, we identified a novel background conductance (I(cat)) in mouse UBSM cells. I(cat) was mediated predominantly by the influx of Na(+), although a small inward Ca(2+) current was detectable with Ca(2+) as the sole cation in the bathing solution. Extracellular Ca(2+), Mg(2+), and Gd(3+) blocked I(cat) in a voltage-dependent manner, with K(i) values at -40 mV of 115, 133, and 1.3 microM, respectively. Although UBSM I(cat) is extensively blocked by physiological extracellular Ca(2+) and Mg(2+), a tonic, depolarizing I(cat) was detected at -40 mV. In addition, inhibition of I(cat) demonstrated a hyperpolarization of the UBSM membrane potential and decreased the amplitude of phasic contractions of isolated UBSM strips. We suggest that I(cat) contributes tonically to the depolarization of the UBSM resting membrane potential, facilitating action potential generation and thereby a maintenance of urinary bladder tone.

Temporal and spatial dimensions of postnatal growth of the mouse urinary bladder urothelium.

Postnatal growth and renewal of mouse urothelium start on the day of birth. In the present study, temporal and spatial dimensions of urothelial growth were studied during the first two postnatal weeks. Quantitative analysis showed that the rate of urothelial cell proliferation is significantly higher during all 14 postnatal days than in adult mice. Three peaks of proliferative and mitotic activity were revealed: on the day of birth and postnatal day 1, on days 6 and 7, and on day 14. The high proliferation rate around the day of birth and at postnatal days 6 and 7 coincides with cell death in the urothelium. Semiquantitative analysis showed that during all 14 postnatal days, the urothelial proliferative response is mostly confined to the basal cell layer. Urothelial cells divide predominantly in parallel to the plain of the urothelium on all chosen postnatal days. Increased portions of urothelial cells, dividing perpendicularly to the urothelium were observed only on the day of birth and on postnatal day 7. Our results suggest that postnatal growth of mouse urothelium is particularly the result of an increasing number of cells in individual cell layers and not the result of an increasing number of cell layers.

Significance of overexpression of metallothionein in mouse urinary bladder focal lesions induced by treatment with N-butyl-N-(4-hydroxybutyl)-nitrosamine.

Metallothionein (MT) is expressed in various types of human tumors, including transitional cell carcinomas of the urinary bladder, but its biological significance remains unclear. In the present study, the role of MT in urinary bladder carcinogenesis induced by N-butyl-N-(4-hydroxybutyl)nitrosamine (BBN) treatment was investigated using C57BL/6 mice. One hundred 5-week-old male C57BL/6 mice were divided into two groups, which were given drinking water with or without 0.05% BBN throughout the experimental period. Subgroups of ten animals from each group were sacrificed at weeks 5, 10, 15, 20 and 25, and urinary bladder samples were examined immunohistochemically for MT, proliferating cell nuclear antigen (PCNA) and apoptosis. MT was found to be abundant in normal-looking mucosa, but decreased with progression from precancerous lesions to invasive carcinoma in the urinary bladder obtained from BBN-treated mice. Lesions could be divided into MT-positive and negative. There was a tendency for greater MT expression in PCNA-positive lesions, while apoptosis was rather associated with MT-negativity. These data suggest that the overexpression of MT may play a role in mouse urinary bladder carcinogenesis.

24 disc Radiation effects in mouse urinary bladder: changes in vascular volume

24 disc Radiation effects in mouse urinary bladder: changes in vascular volume

Radiation dose to abdominal organs of the mouse due to 90Y in the urinary bladder.

Radiation dosimetry estimates in mice have proven useful in evaluating therapeutic radiopharmaceuticals. Current models for mice do not take into account the dose to abdominal organs from radioactivity in the urinary bladder. Although the dose from this source is probably low for slowly clearing compounds such as antibodies, it may be considerable for small molecule (90)Y conjugates undergoing rapid renal clearance. To evaluate this possibility, we modeled the mouse bladder as a 6 mm sphere, surrounded by a 0.5 mm thick shell. We then calculated the radiation dose that might be received by the shell and by more distant points, using the point kernel method with the Loevinger analytical point kernel. A Monte Carlo calculation using EGS4 was also performed. Surface dose calculations were compared with in vitro experimental data. LiF TLD dosimeters were placed directly under five separated, flat-bottomed, 6-mm diameter wells containing (90)Y on a 96-well plate. Dose versus distance from the mouse urinary bladder was calculated using kinetic data from imaging studies of a renally cleared (111)In analog compound currently under investigation. From this, it was estimated that whole body administration of 34.8 MBq of the (90)Y analog compound would yield a bladder wall dose estimate of approximately 98 Gy. Structures within 2 mm of the bladder would receive additional estimated doses of at least 15 Gy. This radiation dose approaches that which is known from external beam data to cause fibrosis in mice. Because of the greater size of the human bladder compared with that of the mouse relative to the range of (90)Y beta particles, the radiation exposure from the same residence time in man was estimated to be considerably lower. This highlights a potential practical limitation of extrapolating radiotoxicity findings in the mouse to human subjects.

Properties and molecular basis of the mouse urinary bladder voltage-gated K+ current.

Potassium channels play an important role in controlling the excitability of urinary bladder smooth muscle (UBSM). Here we describe the biophysical, pharmacological and molecular properties of the mouse UBSM voltage-gated K+ current (IK(V)). The IK(V) activated, deactivated and inactivated slowly with time constants of 29.9 ms at +30 mV, 131 ms at -40 mV and 3.4 s at +20 mV. The midpoints of steady-state activation and inactivation curves were 1.1 mV and -61.4 mV, respectively. These properties suggest that IK(V) plays a role in regulating the resting membrane potential and contributes to the repolarization and after-hyperpolarization phases of action potentials. The IK(V) was blocked by tetraethylammonium ions with an IC50 of 5.2 mM and was unaffected by 1 mM 4-aminopyridine. RT-PCR for voltage-gated K+ channel (KV) subunits revealed the expression of Kv2.1, Kv5.1, Kv6.1, Kv6.2 and Kv6.3 in isolated UBSM myocytes. A comparison of the biophysical properties of UBSM IK(V) with those reported for Kv2.1 and Kv5.1 and/or Kv6 heteromultimeric channels demonstrated a marked similarity. We propose that heteromultimeric channel complexes composed of Kv2.1 and Kv5.1 and/or Kv6 subunits form the molecular basis of the mouse UBSM IK(V).

Neurogenic responses mediated by vanilloid receptor-1 (TRPV1) are blocked by the high affinity antagonist, iodo-resiniferatoxin.

(1) Stimulation of the vanilloid receptor-1 (TRPV1) results in the activation of nociceptive and neurogenic inflammatory responses. Poor specificity and potency of TRPV1 antagonists has, however, limited the clarification of the physiological role of TRPV1. (2) Recently, iodo-resiniferatoxin (I-RTX) has been reported to bind as a high affinity antagonist at the native and heterologously expressed rat TRPV1. Here we have studied the ability of I-RTX to block a series of TRPV1 mediated nociceptive and neurogenic inflammatory responses in different species (including transfected human TRPV1). (3) We have demonstrated that I-RTX inhibited capsaicin-induced mobilization of intracellular Ca(2+) in rat trigeminal neurons (IC(50) 0.87 nM) and in HEK293 cells transfected with the human TRPV1 (IC(50) 0.071 nM). (4) Furthermore, I-RTX significantly inhibited both capsaicin-induced CGRP release from slices of rat dorsal spinal cord (IC(50) 0.27 nM) and contraction of isolated guinea-pig and rat urinary bladder (pK(B) of 10.68 and 9.63, respectively), whilst I-RTX failed to alter the response to high KCl or SP. (5) Finally, in vivo I-RTX significantly inhibited acetic acid-induced writhing in mice (ED(50) 0.42 micro mol kg(-1)) and plasma extravasation in mouse urinary bladder (ED(50) 0.41 micro mol kg(-1)). (6) In in vitro and in vivo TRPV1 activated responses I-RTX was approximately 3 log units and approximately 20 times more potent than capsazepine, respectively. This high affinity antagonist, I-RTX, may be an important tool for future studies in pain and neurogenic inflammatory models.

Heterogeneity of presynaptic muscarinic receptors mediating inhibition of sympathetic transmitter release: a study with M2- and M4-receptor-deficient mice.

1 Presynaptic muscarinic receptors modulate sympathetic transmitter release. The goal of the present study was to identify the muscarinic receptor subtype(s) mediating inhibition of sympathetic transmitter release in mouse atria, urinary bladder and vas deferens. To address this question, electrically evoked noradrenaline release was assessed using tissue preparations from NMRI, M(2)- and M(4)-knockout, and the corresponding M(2)- and M(4)-wildtype mice, after preincubation with (3)H-noradrenaline. 2 The muscarinic agonist carbachol decreased evoked tritium overflow (20 pulses/50 Hz) in each tissue and strain investigated. After deletion of the M(2)-receptor the maximal inhibition by carbachol was significantly reduced (by 41-72%), but not abolished, in all tissues. After deletion of the M(4)-receptor a moderate and significant reduction of the maximal inhibition by carbachol (by 28%) was observed only in the vas deferens. 3 Experiments with the muscarinic antagonists methoctramine and pirenzepine confirmed that the presynaptic muscarinic receptors were predominantly M(2) in atria and bladder and probably a mixture of M(2) and M(4) in the vas deferens. 4 Experiments in the urinary bladder with the cholinesterase inhibitor physostigmine and the muscarinic antagonist ipratropium demonstrated that endogenously released acetylcholine predominantly acted through M(2)-receptors to inhibit noradrenaline release. However, the results do not exclude a minor contribution of M(4)-receptors to this endogenous inhibition. 5 In conclusion, our results clearly indicate that the release-inhibiting muscarinic receptors on postganglionic sympathetic axons in mouse atria, bladder and vas deferens represent mixtures of M(2)- and non-M(2)-receptors. The non-M(2)-receptors remain unknown in atria and the bladder, and may represent primarily M(4)-receptors in the vas deferens. These results reveal an unexpected heterogeneity among the muscarinic receptors mediating inhibition of noradrenaline release.

Activation and sensitisation of low and high threshold afferent fibres mediated by P2X receptors in the mouse urinary bladder.

It has been proposed that extracellular ATP may be involved in visceral mechanosensory transduction by activating ligand-gated ion channels (P2X receptors). In this study, we have investigated the effects of the P2X(3) agonist alpha,beta-methylene ATP (alpha,beta-meATP) and antagonist 2',3'-O-trinitrophenyl-ATP (TNP-ATP) on pelvic afferents innervating the urinary bladder using an in vitro mouse bladder-pelvic nerve preparation. Intravesical application of alpha,beta-meATP (0.03-1 mM) increased multifibre discharges in a concentration-dependent manner. The agonist potentiated, whereas TNP-ATP (0.03 mM) attenuated, the multifibre responses to bladder distensions. Single-unit analysis revealed that both high threshold (HT) fibres (> 15 mmHg; known to be associated with nociception) and low threshold (LT) fibres (< 15 mmHg; probably associated with non-nociceptive events) could be induced to discharge by intravesical alpha,beta-meATP (1 mM, 0.1 ml). The response of the vast majority (21/22, 95.5 %) of HT fibres to bladder distensions was enhanced with a significantly reduced threshold and an increased peak response after exposure to the agonist. On the other hand, 59.7 % (46/77) of LT fibres showed a greater peak and a slightly reduced threshold for response to bladder distension in the presence of alpha,beta-meATP. An additional 11 'silent' fibres became mechanosensitive after exposure to alpha,beta-meATP. TNP-ATP (0.03 mM) did not affect the threshold of LT fibres, but it reduced the peak response of some (22/51, 43.1 %) LT fibres. Conversely, the antagonist resulted in a markedly elevated threshold and reduced peak activity in the majority (13/16, 81.3 %) of HT fibres. The results support the view that P2X(3) receptor-mediated mechanisms contribute to both nociceptive and non-nociceptive (physiological) mechanosensory transduction in the urinary bladder.

Urogenital distribution of a mouse membrane-associated prostaglandin E(2) synthase.

PGE(2) plays a critical role in regulating renal function and facilitating reproduction. One of the rate-limiting biosynthetic enzymes in PGE(2) synthesis is the terminal PGE(2) synthase (PGES). In the present studies, we report the functional expression of a membrane-associated murine PGES (mPGES) and its expression in urogenital tissues. Two independent cDNA clones sharing an identical open reading frame of 459 bp and encoding a peptide of 153 amino acids, but differing in the 3'-untranslated region, were identified. Assays for enzymatic activity, using microsomes prepared from cells transfected with mPGES cDNA, showed that these cDNA sequences encode a functional protein that catalyzes the conversion of PGH(2) to PGE(2). Constitutive expression of mPGES was highest in the mouse kidney, ovary, and urinary bladder but was also expressed at lower levels in uterus and testis. Renal mPGES expression was predominantly localized to epithelia of distal tubules and medullary collecting ducts. High expression was also seen in transitional epithelial cells of bladder and ureter and in the primary and secondary follicles in the ovary. In conclusion, mPGES is constitutively expressed along the urogenital tract, where it may have important roles in normal physiology and disease.

β1‐Subunit of the Ca2+‐activated K+ channel regulates contractile activity of mouse urinary bladder smooth muscle

1. The large-conductance calcium-activated potassium (BK) channel plays an important role in controlling membrane potential and contractility of urinary bladder smooth muscle (UBSM). These channels are composed of a pore-forming alpha-subunit and an accessory, smooth muscle-specific, beta1-subunit. 2. Our aim was to determine the functional role of the beta1-subunit of the BK channel in controlling the contractions of UBSM by using BK channel beta1-subunit 'knock-out' (KO) mice. 3. The beta-galactosidase reporter (lacZ gene) was targeted to the beta1 locus, which provided the opportunity to examine the expression of the beta1-subunit in UBSM. Based on this approach, the beta1-subunit is highly expressed in UBSM. 4. BK channels lacking beta1-subunits have reduced activity, consistent with a shift in BK channel voltage/Ca2+ sensitivity. 5. Iberiotoxin, an inhibitor of BK channels, increased the amplitude and decreased the frequency of phasic contractions of UBSM strips from control mice. 6. The effects of the beta1-subunit deletion on contractions were similar to the effect of iberiotoxin on control mice. The UBSM strips from beta1-subunit KO mice had elevated phasic contraction amplitude and decreased frequency when compared to control UBSM strips. 7. Iberiotoxin increased the amplitude and frequency of phasic contractions, and UBSM tone of UBSM strips from beta1-subunit KO mice, suggesting that BK channels still regulate contractions in the absence of the beta1-subunit. 8. The results indicate that the beta1-subunit, by modulating BK channel activity, plays a significant role in the regulation of phasic contractions of the urinary bladder.

Succession of events in desquamation of superficial urothelial cells as a response to stress induced by prolonged constant illumination

The effect of moderate stress induced by prolonged illumination was analysed on urothelial cells of female mouse urinary bladders at ultrastructural and cytochemical levels. This study demonstrates that the urothelium responds to moderate stress with desquamation which involves two subsequent steps. The first step includes a local detachment of tight junctions and consequently the loss of the permeability barrier leading to expanded intercellular spaces among urothelial cells. During the second step, the disjunction of desmosomes accompanied by exocytosis of lysosomal enzymes (NADPase) in the intercellular space results in exfoliation of superficial cells. It is evident thatmoderate stress elicits an enhanced desquamation of only superficial cells by a subsequent dysfunction of first tight junctions and after that adherens-type junctions. A rapid restoration of the newtight junctions prevents a long-term malfunction of the blood–urine barrier.