Louise Johnston

Calcium oscillations in interstitial cells of the rabbit urethra

Measurements were made (using fast confocal microscopy) of intracellular Ca2+ levels in fluo‐4 loaded interstitial cells isolated from the rabbit urethra. These cells exhibited regular Ca2+ oscillations which were associated with spontaneous transient inward currents recorded under voltage clamp. Interference with d‐myo‐inositol 1,4,5‐trisphosphate (IP3) induced Ca2+ release using 100 μm 2‐aminoethoxydiphenyl borate, and the phospholipase C (PLC) inhibitors 2‐nitro‐4‐carboxyphenyl N,N‐diphenylcarbamate and U73122 decreased the amplitude of spontaneous oscillations but did not abolish them. However, oscillations were abolished when ryanodine receptors were blocked with tetracaine or ryanodine. Oscillations ceased in the absence of external Ca2+, and frequency was directly proportional to the external Ca2+ concentration. Frequency of Ca2+ oscillation was reduced by SKF‐96365, but not by nifedipine. Lanthanum and cadmium completely blocked oscillations. These results suggest that Ca2+ oscillations in isolated rabbit urethral interstitial cells are initiated by Ca2+ release from ryanodine‐sensitive intracellular stores, that oscillation frequency is very sensitive to the external Ca2+ concentration and that conversion of the primary oscillation to a propagated Ca2+ wave depends upon IP3‐induced Ca2+ release.

Morphological expression of KIT positive interstitial cells of Cajal in human bladder.

Purpose We investigated the 3-dimensional morphological arrangement of KIT positive interstitial cells of Cajal in the human bladder and explored their structural interactions with neighboring cells. Materials and Methods Human bladder biopsy samples were prepared for immunohistochemistry/confocal or transmission electron microscopy. Results Whole mount, flat sheet preparations labeled with anti-KIT (Merck, Darmstadt, Germany) contained several immunopositive interstitial cell of Cajal populations. A network of stellate interstitial cells of Cajal in the lamina propria made structural connections with a cholinergic nerve plexus. Vimentin positive cells of several morphologies were present in the lamina propria, presumably including fibroblasts, interstitial cells of Cajal and other cells of mesenchymal origin. Microvessels were abundant in this region and branched, elongated KIT positive interstitial cells of Cajal were found discretely along the vessel axis with each perivascular interstitial cell of Cajal associated with at least 6 vascular smooth muscle cells. Detrusor interstitial cells of Cajal were spindle-shaped, branched cells tracking the smooth muscle bundles, closely associated with smooth muscle cells and vesicular acetylcholine transferase nerves. Rounded, nonbranched KIT positive cells were more numerous in the lamina propria than in the detrusor and were immunopositive for anti-mast cell tryptase. Transmission electron microscopy revealed cells with the ultrastructural characteristics of interstitial cells of Cajal throughout the human bladder wall. Conclusions The human bladder contains a network of KIT positive interstitial cells of Cajal in the lamina propria, which make frequent connections with a cholinergic nerve plexus. Novel perivascular interstitial cells of Cajal were discovered close to vascular smooth muscle cells, suggesting interstitial cells of Cajal-vascular coupling in the bladder. KIT positive detrusor interstitial cells of Cajal tracked smooth muscle bundles and were associated with nerves, perhaps showing a functional tri-unit controlling bladder contractility.

Cholinergic-induced Ca2+ signaling in interstitial cells of Cajal from the guinea pig bladder

Acetylcholine released from parasympathetic excitatory nerves activates contraction in detrusor smooth muscle. Immunohistochemical labeling of guinea pig detrusor with anti-c-Kit and anti-VAChT demonstrated a close structural relationship between interstitial cells of Cajal (ICC) and cholinergic nerves. The ability of guinea pig bladder detrusor ICC to respond to the acetylcholine analog, carbachol, was investigated in enzymatically dissociated cells, loaded with the Ca2+ indicator fluo 4AM. ICC fired Ca2+ transients in response to stimulation by carbachol (1/10 μM). Their pharmacology was consistent with carbachol-induced contractions in strips of detrusor which were inhibited by 4-DAMP (1 μM), an M3 receptor antagonist, but not by the M2 receptor antagonist methoctramine (1 μM). The source of Ca2+ underlying the carbachol transients in isolated ICC was investigated using agents to interfere with influx or release from intracellular stores. Nifedipine (1 μM) or Ni2+ (30–100 μM) to block Ca2+ channels or the removal of external Ca2+ reduced the amplitude of the carbachol transients. Application of ryanodine (30 μM) or tetracaine (100 μM) abolished the transients. The phospholipase C inhibitor, U-73122 (2.5 μM), significantly reduced the responses. 2-Aminoethoxydiethylborate (30 μM) caused a significant reduction and Xestospongin C (1 μM) was more effective, almost abolishing the responses. Intact in situ preparations of guinea pig bladder loaded with a Ca2+ indicator showed distinctively different patterns of spontaneous Ca2+ events in smooth muscle cells and ICC. Both cell types responded to carbachol by an increase in frequency of these events. In conclusion, guinea pig bladder detrusor ICC, both as isolated cells and within whole tissue preparations, respond to cholinergic stimulation by firing Ca2+ transients.

Activation of the cGMP/PKG pathway inhibits electrical activity in rabbit urethral interstitial cells of Cajal by reducing the spatial spread of Ca2+ waves

In the present study we used a combination of patch clamping and fast confocal Ca2+ imaging to examine the effects of activators of the nitric oxide (NO)/cGMP pathway on pacemaker activity in freshly dispersed ICC from the rabbit urethra, using the amphotericin B perforated patch configuration of the patch‐clamp technique. The nitric oxide donor, DEA‐NO, the soluble guanylyl cyclase activator YC‐1 and the membrane‐permeant analogue of cGMP, 8‐Br‐cGMP inhibited spontaneous transient depolarizations (STDs) and spontaneous transient inward currents (STICs) recorded under current‐clamp and voltage‐clamp conditions, respectively. Caffeine‐evoked Cl− currents were unaltered in the presence of SP‐8‐Br‐PET‐cGMPs, suggesting that activation of the cGMP/PKG pathway does not block Cl− channels directly or interfere with Ca2+ release via ryanodine receptors (RyR). However, noradrenaline‐evoked Cl− currents were attenuated by SP‐8‐Br‐PET‐cGMPs, suggesting that activation of cGMP‐dependent protein kinase (PKG) may modulate release of Ca2+ via IP3 receptors (IP3R).When urethral interstitial cells (ICC) were loaded with Fluo4‐AM (2 μm), and viewed with a confocal microscope, they fired regular propagating Ca2+ waves, which originated in one or more regions of the cell. Application of DEA‐NO or other activators of the cGMP/PKG pathway did not significantly affect the oscillation frequency of these cells, but did significantly reduce their spatial spread. These effects were mimicked by the IP3R blocker, 2‐APB (100 μm). These data suggest that NO donors and activators of the cGMP pathway inhibit electrical activity of urethral ICC by reducing the spatial spread of Ca2+ waves, rather than decreasing wave frequency.

Contribution of reverse Na+–Ca2+ exchange to spontaneous activity in interstitial cells of Cajal in the rabbit urethra

Interstitial cells of Cajal (ICC) isolated from the rabbit urethra exhibit regular Ca2+ oscillations that are associated with spontaneous transient inward currents (STICs) recorded under voltage clamp. Their frequency is known to be very sensitive to external Ca2+ concentration but the mechanism of this has yet to be elucidated. In the present study experiments were performed to assess the role of Na+–Ca2+ exchange (NCX) in this process. Membrane currents were recorded using the patch clamp technique and measurements of intracellular Ca2+ were made using fast confocal microscopy. When reverse mode NCX was enhanced by decreasing the external Na+ concentration [Na+]o from 130 to 13 mm, the frequency of global Ca2+ oscillations and STICs increased. Conversely, inhibition of reverse mode NCX by KB‐R7943 and SEA0400 decreased the frequency of Ca2+ oscillations and STICs. Application of caffeine (10 mm) and noradrenaline (10 μm) induced transient Ca2+‐activated chloride currents (IClCa) at −60 mV due to release of Ca2+ from ryanodine‐ and inositol trisphosphate (IP3)‐sensitive Ca2+ stores, respectively, but these responses were not blocked by KB‐R7943 or SEA0400 suggesting that neither drug blocked Ca2+‐activated chloride channels or Ca2+ release from stores. Intact strips of rabbit urethra smooth muscle develop spontaneous myogenic tone. This tone was relaxed by application of SEA0400 in a concentration‐dependent fashion. Finally, single cell RT‐PCR experiments revealed that isolated ICC from the rabbit urethra only express the type 3 isoform of the Na+–Ca2+ exchanger (NCX3). These results suggest that frequency of spontaneous activity in urethral ICC can be modulated by Ca2+ entry via reverse NCX.

Identification of PDGFRα Positive Populations of Interstitial Cells in Human and Guinea Pig Bladders

PURPOSE The bladder wall comprises a complex array of cells, including urothelium, smooth muscle, nerves and interstitial cells. Interstitial cells have several subtypes based on site, morphology and differential expression of markers such as anti-vimentin and anti-KIT. We examined whether a subpopulation of interstitial cells immunopositive for PDGFRα exists in human and guinea pig bladders. MATERIALS AND METHODS Human and guinea pig bladder tissues were processed for immunohistochemistry and examined by bright field or confocal microscopy. Whole mount tissues and paraffin sections were labeled with antibodies to PDGFRα, vimentin, KIT and PGP9.5. Protein expression was assessed by Western blot. RESULTS PDGFRα(+) cells were present in human and guinea pig bladders. In the guinea pig PDGFRα(+) cells had a branched stellate morphology and formed networks in the lamina propria. In human and guinea pig detrusors PDGFRα(+) cells were elongated on the boundary of smooth muscle bundles or were seen as groups of stellate cells in the interbundle spaces. PDGFRα(+) cells were located close to nerves labeled by PGP9.5. Double labeling revealed that PDGFRα(+) cells were a subgroup of the vimentin(+) population. A significant proportion of PDGFRα(+) cells were also KIT(+). Bands corresponding to PDGFRα, KIT and vimentin proteins were detected on Western blot. CONCLUSIONS To our knowledge this study is the first to identify PDGFRα(+)/KIT(+) cells in the bladder lamina propria and detrusor layers. These cells are a subgroup of the vimentin(+) population, showing the complexity of bladder interstitial cells. PDGFRα(+) cells are apparently structurally associated with intramural nerves, indicating integration with bladder control mechanisms.

Altered distribution of interstitial cells and innervation in the rat urinary bladder following spinal cord injury.

Changes in the distribution of interstitial cells (IC) are reportedly associated with dysfunctional bladder. This study investigated whether spinal cord injury (SCI) resulted in changes to IC subpopulations (vimentin‐positive with the ultrastructural profile of IC), smooth muscle and nerves within the bladder wall and correlated cellular remodelling with functional properties. Bladders from SCI (T8/9 transection) and sham‐operated rats 5 weeks post‐injury were used for ex vivo pressure–volume experiments or processed for morphological analysis with transmission electron microscopy (TEM) and light/confocal microscopy. Pressure‐volume relationships revealed low‐pressure, hypercompliance in SCI bladders indicative of decompensation. Extensive networks of vimentin‐positive IC were typical in sham lamina propria and detrusor but were markedly reduced post‐SCI; semi‐quantitative analysis showed significant reduction. Nerves labelled with anti‐neurofilament and anti‐vAChT were notably decreased post‐SCI. TEM revealed lamina propria IC and detrusor IC which formed close synaptic‐like contacts with vesicle‐containing nerve varicosities in shams. Lamina propria and detrusor IC were ultrastructurally damaged post‐SCI with retracted/lost cell processes and were adjacent to areas of cellular debris and neuronal degradation. Smooth muscle hypertrophy was common to SCI tissues. In conclusion, IC populations in bladder wall were decreased 5 weeks post‐SCI, accompanied with reduced innervation, smooth muscle hypertrophy and increased compliance. These novel findings indicate that bladder wall remodelling post‐SCI affects the integrity of interactions between smooth muscle, nerves and IC, with compromised IC populations. Correlation between IC reduction and a hypercompliant phenotype suggests that disruption to bladder IC contribute to pathophysiological processes underpinning the dysfunctional SCI bladder.

The passive and active contractile properties of the neurogenic, underactive bladder.

Detrusor underactivity is highly prevalent, particularly in the elderly. It is assumed to result from detrusor failure, although detrusor contractility is often derived from urodynamics studies. Given that detrusor pressure and force are not proportional and urodynamics cannot identify the basis of the pathology, we produced a neurogenic animal model with a highly‐compliant bladder and studied detrusor muscle properties, aiming to increase our understanding of the underlying pathology. Highly compliant bladders were characterized by reduced passive wall stiffness and stretched detrusor muscle strips exhibited an enhanced rate of relaxation. These detrusor strips displayed spontaneous contractions that were of greater amplitude (expressed as a ratio of bladder wall stiffness) than those of strips from sham‐operated animals; spontaneous contractions increased in amplitude when stimulated by an agonist. These data imply that compliance is not the result of a reduction of detrusor contractility; we hypothesize that altered matrix properties reduce the magnitude with which force can be generated to void the bladder.

Calbindin 2 (CALB2) Regulates 5-Fluorouracil Sensitivity in Colorectal Cancer by Modulating the Intrinsic Apoptotic Pathway

The role of the calcium binding protein, Calbindin 2 (CALB2), in regulating the response of colorectal cancer (CRC) cells to 5-Fluorouracil (5-FU) was investigated. Real-time RT-PCR and Western blot analysis revealed that CALB2 mRNA and protein expression were down-regulated in p53 wild-type and p53 null isogenic HCT116 CRC cell lines following 48 h and 72 h 5-FU treatment. Moreover, 5-FU-induced apoptosis was significantly reduced in HCT116 and LS174T CRC cell lines in which CALB2 expression had been silenced. Further investigation revealed that CALB2 translocated to the mitochondria following 5-FU treatment and that 5-FU-induced loss of mitochondrial membrane potential (Δψm) was abrogated in CALB2-silenced cells. Furthermore, CALB2 silencing decreased 5-FU-induced cytochrome c and smac release from the mitochondria and also decreased 5-FU-induced activation of caspases 9 and 3/7. Of note, co-silencing of XIAP overcame 5-FU resistance in CALB2-silenced cells. Collectively, these results suggest that following 5-FU treatment in CRC cell lines, CALB2 is involved in apoptosis induction through the intrinsic mitochondrial pathway. This indicates that CALB2 may be an important mediator of 5-FU-induced cell death. Moreover, down-regulation of CALB2 in response to 5-FU may represent an intrinsic mechanism of resistance to this anti-cancer drug.

The physiological properties of the decompensated, spinal cord injured bladder

Hypothesis / aims of study Conditional electrical stimulation of the dorsal genital nerve, applied at the onset of an involuntary detrusor contraction can suppress the involuntary contraction [1]. This both maintains low storage pressure and prevents incontinence episodes. The aim of this study was to investigate whether an implantable pressure sensor placed in the bladder wall could be used to detect the onset of bladder contractions.