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Paediatric acute myeloid leukemia (AML) is a heterogeneous hematological malignancy still heavily reliant on traditional chemotherapeutic approaches. Combination treatments have shown to be a superior approach, but their success is often hindered by side effects and different drugs' pharmacokinetics. Here, we investigated ABT-737 and Purvalanol A as a potential drug pairing for pediatric AML and described the development of CD33-targeted polymeric nanoparticles (NPs) to enable their simultaneous targeted codelivery. Separate drug encapsulation within poly(lactic- co -glycolic acid) (PLGA) NPs was optimized prior to coencapsulation of both drugs at a synergistic ratio in PEGylated PLGA NPs. The therapeutic effects of formulations were evaluated in a panel of pediatric AML cells, and dual drug-loaded NPs (dual NPs) demonstrated significantly enhanced apoptotic cell death. Moreover, conjugation to gemtuzumab resulted in improved NP binding and internalization in CD33-positive cells. Finally, CD33-targeted dual-loaded NPs showed enhanced cytotoxicity to CD33-positive AML cells via CD33-mediated targeted drug delivery.

Introduction: Urofacial, or Ochoa, syndrome (UFS) is an autosomal recessive disease featuring a dyssynergic bladder with detrusor smooth muscle contracting against an undilated outflow tract. It also features an abnormal grimace. Half of individuals with UFS carry biallelic variants in HPSE2 , whereas other rare families carry variants in LRIG2. LRIG2 is immunodetected in pelvic ganglia sending autonomic axons into the bladder. Moreover, Lrig2 mutant mice have abnormal urination and abnormally patterned bladder nerves. We hypothesized that peripheral neurogenic defects underlie LRIG2 -associated bladder dysfunction., Methods: We describe a new family with LRIG2 -associated UFS and studied Lrig2 homozygous mutant mice with ex vivo physiological analyses., Results: The index case presented antenatally with urinary tract (UT) dilatation, and postnatally had urosepsis and functional bladder outlet obstruction. He had the grimace that, together with UT disease, characterizes UFS. Although HPSE2 sequencing was normal, he carried a homozygous, predicted pathogenic, LRIG2 stop variant (c.1939C>T; p.Arg647∗). Lrig2 mutant mice had enlarged bladders. Ex vivo physiology experiments showed neurogenic smooth muscle relaxation defects in the outflow tract, containing the urethra adjoining the bladder, and in detrusor contractility. Moreover, there were nuanced differences in physiological outflow tract defects between the sexes., Conclusion: Putting this family in the context of all reported UT disease-associated LRIG2 variants, the full UFS phenotype occurs with biallelic stop or frameshift variants, but missense variants lead to bladder-limited disease. Our murine observations support the hypothesis that UFS is a genetic autonomic neuropathy of the bladder affecting outflow tract and bladder body function., (© 2023 International Society of Nephrology. Published by Elsevier Inc.)

In addition, to their established role in cardiac myocytes and neurons, ion channels encoded by ether-a-go-go-related genes (ERG1-3 or kcnh2,3 and 6) (kcnh2) are functionally relevant in phasic smooth muscle. The aim of the study was to determine the expression and functional impact of ERG expression products in rat urinary bladder smooth muscle using quantitative polymerase chain reaction, immunocytochemistry, whole-cell patch-clamp and isometric tension recording. kcnh2 was expressed in rat bladder, whereas kcnh6 and kcnh3 expression were negligible. Immunofluorescence for the kcnh2 expression product Kv11.1 was detected in the membrane of isolated smooth muscle cells. Potassium currents with voltage-dependent characteristics consistent with Kv11.1 channels and sensitive to the specific blocker E4031 (1 μM) were recorded from isolated detrusor smooth muscles. Disabling Kv11.1 activity with specific blockers (E4031 and dofetilide, 0.2-20 μM) augmented spontaneous contractions to a greater extent than BK Ca channel blockers, enhanced carbachol-driven activity, increased nerve stimulation-mediated contractions, and impaired β-adrenoceptor-mediated inhibitory responses. These data establish for the first time that Kv11.1 channels are key determinants of contractility in rat detrusor smooth muscle., (© 2023 The Authors. Physiological Reports published by Wiley Periodicals LLC on behalf of The Physiological Society and the American Physiological Society.)

Knowledge of the participation of ATP and related purines in urinary tract physiology has been established over the last five decades through the work of many independent groups, inspired by, and building on the pioneering studies of Professor Geoffrey Burnstock and his coworkers. As part of a series of reviews in this tribute edition, the present article summarises our current understanding of purines and purinergic signalling in modulating and regulating urinary tract function. Purinergic mechanisms underlying the origin of bladder pain; sensations of bladder filling and urinary tract motility; and regulation of detrusor smooth muscle contraction are described, encompassing the relevant history of discovery and consolidation of knowledge as methodologies and pharmacological tools have developed. We consider normal physiology, including development and ageing and then move to pathophysiology, discussing the causal and consequential contribution of purinergic signalling mechanism and their constituent components (receptors, signal transduction, effector molecules) to bladder dysfunction., (Copyright © 2021 Elsevier B.V. All rights reserved.)

Objective(s): Interpersonal problems are one of the most persistent difficulties facing those with personality disorders (PDs) and are linked with dysfunction across numerous social domains. Using an interpersonal model of PDs, we examined the indirect effects of Avoidant PD (AvPD) symptoms and social dysfunction through interpersonal problems, as well as Borderline PD (BPD) symptoms and social dysfunction., Methods: Participants were 226 adults taking part in an outpatient treatment program., Results: Using cross-sectional data from self-reported measures, we found that cold (b = 0.10, 95% confidence interval (CI) [0.038, 0.176]) and overly nurturant (b = 0.04, 95% CI [0.001, 0.090]) interpersonal problems showed an indirect association between AvPD symptoms and social dysfunction. The only significant indirect association between BPD symptoms and social dysfunction was overly nurturant (b = 0.05, 95% CI [0.001, 0.120])., Conclusions: Results may aid in the development of more individualized treatments for AvPD and BPD symptoms., (© 2020 Wiley Periodicals LLC.)

Aims: Urofacial syndrome (UFS) is an autosomal recessive disease characterized by detrusor contraction against an incompletely dilated outflow tract. This dyssynergia causes dribbling incontinence and incomplete voiding. Around half of individuals with UFS have biallelic mutations of HPSE2 that encodes heparanase 2, a protein found in pelvic ganglia and bladder nerves. Homozygous Hpse2 mutant mice have abnormal patterns of nerves in the bladder body and outflow tract, and also have dysfunctional urinary voiding. We hypothesized that bladder neurophysiology is abnormal Hpse2 mutant mice., Methods: Myography was used to study bladder bodies and outflow tracts isolated from juvenile mice. Myogenic function was analyzed after chemical stimulation or blockade of key receptors. Neurogenic function was assessed by electrical field stimulation (EFS). Muscarinic receptor expression was semi-quantified by Western blot analysis., Results: Nitrergic nerve-mediated relaxation of precontracted mutant outflow tracts was significantly decreased vs littermate controls. The contractile ability of mutant outflow tracts was normal as assessed by KCl and the α1-adrenoceptor agonist phenylephrine. EFS of mutant bladder bodies induced significantly weaker contractions than controls. Conversely, the muscarinic agonist carbachol induced significantly stronger contractions of bladder body than controls., Conclusions: The Hpse2 model of UFS features aberrant bladder neuromuscular physiology. Further work is required to determine whether similar aberrations occur in patients with UFS., (© 2020 The Authors. Neurourology and Urodynamics Published by Wiley Periodicals LLC.)

Background: While there is some evidence that patients with elevated depression symptoms experience greater benefits from negative-affect expression, this amplifying effect has not been investigated within a psychotherapeutic context. This is particularly notable considering the substantial evidence linking affect expression with desired outcomes across psychotherapeutic approaches. The primary purpose of the present study was to investigate if depression symptoms amplified (i.e., moderated) the association between negative-affect expression and reductions in interpersonal problems for patients in a psychiatric day treatment for personality disorders. The secondary purpose was to investigate milieu (i.e., other patients concurrently enrolled) negative-affect intensity and its association with patients' reductions in interpersonal problems., Methods: Participants (N = 239) were patients consecutively admitted into a psychiatric day treatment program for people with personality disorders. Patients completed measures of (1) depression symptoms at pre-treatment, (2) interpersonal problems at pre- and post-treatment, and (3) negative-affect expression each week of treatment., Results: The association between patient negative-affect expression and reductions in interpersonal problems was amplified by depression symptoms. Depression symptoms also amplified the association between milieu negative-affect intensity and reductions in interpersonal problems., Conclusions: Our findings indicate that depression symptoms amplified the therapeutic value of negative-affect expression and amplified the value of an affectively intense milieu. Practical and theoretical implications are discussed., Competing Interests: Declaration of Competing Interest All authors declare no conflicts of interest., (Copyright © 2020 Elsevier B.V. All rights reserved.)

Several studies indicate that pelvic ischemia and oxidative stress may play a significant role in lower urinary tract dysfunction (LUTD), including detrusor overactivity (DO)/overactive bladder (OAB) and detrusor underactivity (DU)/underactive bladder (UAB). The present article addresses proposal 1: "Are oxidative stress and ischemia significant causes of bladder damage leading to LUTD?" from the 2019 International Consultation on Incontinence-Research Society (ICI-RS) meeting. Bladder ischemia in animals and humans is briefly described, along with the proposed progression from ischemia to LUTD. Bladder ischemia is compared with ischemia of other organs, and the ongoing development of pelvic ischemia animal models is discussed. In addition, the distribution of blood within the bladder during filling and voiding and the challenges of quantification of blood flow in vivo are described. Furthermore, oxidative stress, including potential biomarkers and treatments, and challenges regarding antioxidant therapy for the treatment of LUTD are discussed. Finally, seven critical research questions and proposed studies to answer those questions were identified as priorities that would lead to major advances in the understanding and treatment of lower urinary tract symptoms (LUTS)/LUTD associated with pelvic ischemia and oxidative stress., (© 2020 Wiley Periodicals, Inc.)

Aims: This article reviews current knowledge of the underpinning mechanisms of how the bladder senses fullness locally and also revisits clinical measurements of lower urinary tract sensation. The former represents cellular sensing during bladder filling whereas the latter describes the sensations leading to conscious perception of bladder fullness., Methods: The topic was discussed in a "think tank" session at the 2019 International Consultation on Incontinence-Research Symposium in Bristol, UK; summarized in the present review., Results: Recent advances in the basic science of bladder sensing relating to (a) the bladder wall-urothelial cells, sensory nerves, interstitial cells, and smooth muscle cells and (b) putative chemo/mechanosensors in the urethra-paraneurons or "brush cells" are discussed. Validated clinical measurement of lower urinary tract sensation is reviewed in the context of how this could be better harnessed for patient benefit. We discuss the potential of app/tablet/mobile technology based on triggers and distractors to override aberrant local sensing/higher sensation and how these technologies could be utilized in treatment., Conclusions: We conclude that a better understanding of bladder sensation is essential to inform clinical management of lower urinary tract symptoms., (© 2020 Wiley Periodicals, Inc.)

The urinary bladder has two functions: to store urine, when it is relaxed and highly compliant; and void its contents, when intravesical pressure rises due to co-ordinated contraction of detrusor smooth muscle in the bladder wall. Superimposed on this description are two observations: (1) the normal, relaxed bladder develops small transient increases of intravesical pressure, mirrored by local bladder wall movements; (2) pathological, larger pressure variations (detrusor overactivity) can occur that may cause involuntary urine loss and/or detrusor overactivity. Characterisation of these spontaneous contractions is important to understand: how normal bladder compliance is maintained during filling; and the pathophysiology of detrusor overactivity. Consideration of how spontaneous contractions originate should include the structural complexity of the bladder wall. Detrusor smooth muscle layer is overlain by a mucosa, itself a complex structure of urothelium and a lamina propria containing sensory nerves, micro-vasculature, interstitial cells and diffuse muscular elements.Several theories, not mutually exclusive, have been advanced for the origin of spontaneous contractions. These include intrinsic rhythmicity of detrusor muscle; modulation by non-muscular pacemaking cells in the bladder wall; motor input to detrusor by autonomic nerves; regulation of detrusor muscle excitability and contractility by the adjacent mucosa and spontaneous contraction of elements of the lamina propria. This chapter will consider evidence for each theory in both normal and overactive bladder and how their significance may vary during ageing and development. Further understanding of these mechanisms may also identify novel drug targets to ameliorate the clinical consequences of large contractions associated with detrusor overactivity.

Introduction: Storage phase bladder activity is a counter-intuitive observation of spontaneous contractions. They are potentially an intrinsic feature of the smooth muscle, but interstitial cells in the mucosa and the detrusor itself, as well as other muscular elements in the mucosa may substantially influence them. They are identified in several models explaining lower urinary tract dysfunction., Methods: A consensus meeting at the International Consultation on Incontinence Research Society (ICI-RS) 2017 congress considered the origins and relevance of spontaneous bladder contractions by debating which cell type(s) modulate bladder spontaneous activity, whether the methodologies are sufficiently robust, and implications for healthy and abnormal lower urinary tract function., Results: The identified research priorities reflect a wide range of unknown aspects. Cellular contributions to spontaneous contractions in detrusor smooth muscle are still uncertain. Accordingly, insight into the cellular physiology of the bladder wall, particularly smooth muscle cells, interstitial cells, and urothelium, remains important. Upstream influences, such as innervation, endocrine, and paracrine factors, are particularly important. The cellular interactions represent the key understanding to derive the integrative physiology of organ function, notably the nature of signalling between mucosa and detrusor layers. Indeed, it is still not clear to what extent spontaneous contractions generated in isolated preparations mirror their normal and pathological counterparts in the intact bladder. Improved models of how spontaneous contractions influence pressure generation and sensory nerve function are also needed., Conclusions: Deriving approaches to robust evaluation of spontaneous contractions and their influences for experimental and clinical use could yield considerable progress in functional urology., (© 2018 Wiley Periodicals, Inc.)

Radiation-induced bladder toxicity is associated with radiation therapy for pelvic malignancies, arising from unavoidable irradiation of neighbouring normal bladder tissue. This study aimed to investigate the acute impact of ionizing radiation on the contractility of bladder strips and identify the radiation-sensitivity of the mucosa vs the detrusor. Guinea-pig bladder strips (intact or mucosa-free) received ex vivo sham or 20Gy irradiation and were studied with in vitro myography, electrical field stimulation and Ca2+-fluorescence imaging. Frequency-dependent, neurogenic contractions in intact strips were reduced by irradiation across the force-frequency graph. The radiation-difference persisted in atropine (1μM); subsequent addition of PPADs (100μM) blocked the radiation effect at higher stimulation frequencies and decreased the force-frequency plot. Conversely, neurogenic contractions in mucosa-free strips were radiation-insensitive. Radiation did not affect agonist-evoked contractions (1μM carbachol, 5mM ATP) in intact or mucosa-free strips. Interestingly, agonist-evoked contractions were larger in irradiated mucosa-free strips vs irradiated intact strips suggesting that radiation may have unmasked an inhibitory mucosal element. Spontaneous activity was larger in control intact vs mucosa-free preparations; this difference was absent in irradiated strips. Spontaneous Ca2+-transients in smooth muscle cells within tissue preparations were reduced by radiation. Radiation affected neurogenic and agonist-evoked bladder contractions and also reduced Ca2+-signalling events in smooth muscle cells when the mucosal layer was present. Radiation eliminated a positive modulatory effect on spontaneous activity by the mucosa layer. Overall, the findings suggest that radiation impairs contractility via mucosal regulatory mechanisms independent of the development of radiation cystitis.

Radiation therapy (RT) delivers tumour kill, directly and often via bystander mechanisms. Bladder toxicity is a dose limiting constraint in pelvic RT, manifested as radiation cystitis and urinary symptoms. We aimed to investigate the impact of radiation-induced bystander signaling on normal/cancer urothelial cells. Human urothelial cancer cells T24, HT1376 and normal urothelial cells HUC, SV-HUC were used. Cells were irradiated and studied directly, or conditioned medium from irradiated cells (CM) was transferred to naïve, cells. T24 or SV-HUC cells in the shielded half of irradiated flasks had increased numbers of DNA damage foci vs non-irradiated cells. A physical barrier blocked this response, indicating release of transmitters from irradiated cells. Clonogenic survival of shielded T24 or SV-HUC was also reduced; a physical barrier prevented this phenomenon. CM-transfer increased pro-apoptotic caspase-3 activity, increased cleaved caspase-3 and cleaved PARP expression and reduced survival protein XIAP expression. This effect was mimicked by ATP. ATP or CM evoked suramin-sensitive Ca 2+ -signals. Irradiation increased [ATP] in CM from T24. The CM-inhibitory effect on T24 clonogenic survival was blocked by apyrase, or mimicked by ATP. We conclude that radiation-induced bystander signaling enhances urothelial cancer cell killing via activation of purinergic pro-apoptotic pathways. This benefit is accompanied by normal urothelial damage indicating RT bladder toxicity is also bystander-mediated., Competing Interests: CONFLICTS OF INTEREST The authors declare that there are no conflicts of interest.

Aims: To investigate the local, regulatory role of the mucosa on bladder strip contractility from normal and overactive bladders and to examine the effect of botulinum toxin A (BoNT-A)., Methods: Bladder strips from spontaneously hyperactive rat (SHR) or normal rats (Sprague Dawley, SD) were dissected for myography as intact or mucosa-free preparations. Spontaneous, neurogenic and agonist-evoked contractions were investigated. SHR strips were incubated in BoNT-A (3 h) to assess effects on contractility., Results: Spontaneous contraction amplitude, force-integral or frequency were not significantly different in SHR mucosa-free strips compared with intacts. In contrast, spontaneous contraction amplitude and force-integral were smaller in SD mucosa-free strips than in intacts; frequency was not affected by the mucosa. Frequency of spontaneous contractions in SHR strips was significantly greater than in SD strips. Neurogenic contractions in mucosa-free SHR and SD strips at higher frequencies were smaller than in intact strips. The mucosa did not affect carbachol-evoked contractions in intact versus mucosa-free strips from SHR or SD bladders. BoNT-A reduced spontaneous contractions in SHR intact strips; this trend was also observed in mucosa-free strips but was not significant. Neurogenic and carbachol-evoked contractions were reduced by BoNT-A in mucosa-free but not intact strips. Depolarisation-induced contractions were smaller in BoNT-A-treated mucosa-free strips., Conclusions: The mucosal layer positively modulates spontaneous contractions in strips from normal SD but not overactive SHR bladder strips. The novel finding of BoNT-A reduction of contractions in SHR mucosa-free strips indicates actions on the detrusor, independent of its classical action on neuronal SNARE complexes., (© 2016 Wiley Periodicals, Inc.)

Aims: This article summarizes discussion at the International Consultation on Incontinence Research Society (ICI-RS) 2015 meeting of urine modification in the urinary tract by the urothelium. It considers the literature and proposes pertinent questions that need to be addressed to understand this phenomenon within a physiological context., Methods: Following the ICI-RS meeting, publications in PubMed relating to urine modification in the renal pelvis, ureter, and bladder were reviewed., Results: Historically, the urothelium has been simply considered as a passive, impermeable barrier, preventing contact between urine and the underlying cells. In addition to the ability of the umbrella cells to modify the surface area of the urothelium during bladder filling, the urothelium may also be involved in modifying urine composition. Several lines of evidence support the hypothesis that electrolytes and water can be reabsorbed by the urothelium and that this may have physiological relevance. Firstly, urothelial cells express several types of aquaporins and ion channels; the membrane expression of which is modulated by the extracellular concentration of ions including Na + . Secondly, studies of urine composition in the renal pelvis and bladder demonstrate urine modification, indicating that water and/or electrolyte transport has occurred. Thirdly, hibernating mammals, with urothelial and bladder wall histology similar to non-hibernating mammals are known to produce and reabsorb urine daily, during long periods of hibernation., Conclusions: The phenomenon of urine modification by the urothelium may be physiologically important during normal bladder filling. Research should be focused on investigating how this may change in conditions of urinary dysfunction., (© 2017 Wiley Periodicals, Inc.)

Background: Calcium channel blockers (CCBs) may affect prostate cancer (PCa) growth by various mechanisms including those related to androgens. The fusion of the androgen-regulated gene TMPRSS2 and the oncogene ERG (TMPRSS2:ERG or T2E) is common in PCa, and prostate tumors that harbor the gene fusion are believed to represent a distinct disease subtype. We studied the association of CCB use with the risk of PCa, and molecular subtypes of PCa defined by T2E status., Methods: Participants were residents of King County, Washington, recruited for population-based case-control studies (1993-1996 or 2002-2005). Tumor T2E status was determined by fluorescence in situ hybridization using tumor tissue specimens from radical prostatectomy. Detailed information on use of CCBs and other variables was obtained through in-person interviews. Binomial and polytomous logistic regression were used to generate odds ratios (ORs) and 95% confidence intervals (CIs)., Results: The study included 1,747 PCa patients and 1,635 age-matched controls. A subset of 563 patients treated with radical prostatectomy had T2E status determined, of which 295 were T2E positive (52%). Use of CCBs (ever vs. never) was not associated with overall PCa risk. However, among European-American men, users had a reduced risk of higher-grade PCa (Gleason scores ≥7: adjusted OR = 0.64; 95% CI: 0.44-0.95). Further, use of CCBs was associated with a reduced risk of T2E positive PCa (adjusted OR = 0.38; 95% CI: 0.19-0.78), but was not associated with T2E negative PCa., Conclusions: This study found suggestive evidence that use of CCBs is associated with reduced relative risks for higher Gleason score and T2E positive PCa. Future studies of PCa etiology should consider etiologic heterogeneity as PCa subtypes may develop through different causal pathways. Prostate 77:282-290, 2017. © 2016 Wiley Periodicals, Inc., (© 2016 Wiley Periodicals, Inc.)

Background: Previously we identified a DNA damage response-deficient (DDRD) molecular subtype within breast cancer. A 44-gene assay identifying this subtype was validated as predicting benefit from DNA-damaging chemotherapy. This subtype was defined by interferon signaling. In this study, we address the mechanism of this immune response and its possible clinical significance., Methods: We used immunohistochemistry (IHC) to characterize immune infiltration in 184 breast cancer samples, of which 65 were within the DDRD subtype. Isogenic cell lines, which represent DDRD-positive and -negative, were used to study the effects of chemokine release on peripheral blood mononuclear cell (PBMC) migration and the mechanism of immune signaling activation. Finally, we studied the association between the DDRD subtype and expression of the immune-checkpoint protein PD-L1 as detected by IHC. All statistical tests were two-sided., Results: We found that DDRD breast tumors were associated with CD4+ and CD8+ lymphocytic infiltration (Fisher's exact test P < .001) and that DDRD cells expressed the chemokines CXCL10 and CCL5 3.5- to 11.9-fold more than DNA damage response-proficient cells (P < .01). Conditioned medium from DDRD cells statistically significantly attracted PBMCs when compared with medium from DNA damage response-proficient cells (P < .05), and this was dependent on CXCL10 and CCL5. DDRD cells demonstrated increased cytosolic DNA and constitutive activation of the viral response cGAS/STING/TBK1/IRF3 pathway. Importantly, this pathway was activated in a cell cycle-specific manner. Finally, we demonstrated that S-phase DNA damage activated expression of PD-L1 in a STING-dependent manner., Conclusions: We propose a novel mechanism of immune infiltration in DDRD tumors, independent of neoantigen production. Activation of this pathway and associated PD-L1 expression may explain the paradoxical lack of T-cell-mediated cytotoxicity observed in DDRD tumors. We provide a rationale for exploration of DDRD in the stratification of patients for immune checkpoint-based therapies., (© The Author 2016. Published by Oxford University Press. All rights reserved. For Permissions, please email: journals.permissions@oup.com.)

The importance of ion channels in the hallmarks of many cancers is increasingly recognised. This article reviews current knowledge of the expression of members of the voltage-gated calcium channel family (Ca V ) in cancer at the gene and protein level and discusses their potential functional roles. The ten members of the Ca V channel family are classified according to expression of their pore-forming α-subunit; moreover, co-expression of accessory α2δ, β and γ confers a spectrum of biophysical characteristics including voltage dependence of activation and inactivation, current amplitude and activation/inactivation kinetics. Ca V channels have traditionally been studied in excitable cells including neurones, smooth muscle, skeletal muscle and cardiac cells, and drugs targeting the channels are used in the treatment of hypertension and epilepsy. There is emerging evidence that several Ca V channels are differentially expressed in cancer cells compared to their normal counterparts. Interestingly, a number of Ca V channels also have non-canonical functions and are involved in transcriptional regulation of the expression of other proteins including potassium channels. Pharmacological studies show that Ca V canonical function contributes to the fundamental biology of proliferation, cell-cycle progression and apoptosis. This raises the intriguing possibility that calcium channel blockers, approved for the treatment of other conditions, could be repurposed to treat particular cancers. Further research will reveal the full extent of both the canonical and non-canonical functions of Ca V channels in cancer and whether calcium channel blockers are beneficial in cancer treatment.

Development of cribriform morphology (CM) heralds malignant change in human colon but lack of mechanistic understanding hampers preventive therapy. This study investigated CM pathobiology in three-dimensional (3D) Caco-2 culture models of colorectal glandular architecture, assessed translational relevance and tested effects of 1,25(OH)2D3,theactive form of vitamin D. CM evolution was driven by oncogenic perturbation of the apical polarity (AP) complex comprising PTEN, CDC42 and PRKCZ (phosphatase and tensin homolog, cell division cycle 42 and protein kinase C zeta). Suppression of AP genes initiated a spatiotemporal cascade of mitotic spindle misorientation, apical membrane misalignment and aberrant epithelial configuration. Collectively, these events promoted "Swiss cheese-like" cribriform morphology (CM) comprising multiple abnormal "back to back" lumens surrounded by atypical stratified epithelium, in 3D colorectal gland models. Intestinal cancer driven purely by PTEN-deficiency in transgenic mice developed CM and in human CRC, CM associated with PTEN and PRKCZ readouts. Treatment of PTEN-deficient 3D cultures with 1,25(OH)2D3 upregulated PTEN, rapidly activated CDC42 and PRKCZ, corrected mitotic spindle alignment and suppressed CM development. Conversely, mutationally-activated KRAS blocked1,25(OH)2D3 rescue of glandular architecture. We conclude that 1,25(OH)2D3 upregulates AP signalling to reverse CM in a KRAS wild type (wt), clinically predictive CRC model system. Vitamin D could be developed as therapy to suppress inception or progression of a subset of colorectal tumors., Competing Interests: None.

Neurogenic detrusor overactivity (NDO) is a well known consequence of spinal cord injury (SCI), recognizable after spinal shock, during which the bladder is areflexic. NDO emergence and maintenance depend on profound plastic changes of the spinal neuronal pathways regulating bladder function. It is well known that neurotrophins (NTs) are major regulators of such changes. NGF is the best-studied NT in the bladder and its role in NDO has already been established. Another very abundant neurotrophin is BDNF. Despite being shown that, acting at the spinal cord level, BDNF is a key mediator of bladder dysfunction and pain during cystitis, it is presently unclear if it is also important for NDO. This study aimed to clarify this issue. Results obtained pinpoint BDNF as an important regulator of NDO appearance and maintenance. Spinal BDNF expression increased in a time-dependent manner together with NDO emergence. In chronic SCI rats, BDNF sequestration improved bladder function, indicating that, at later stages, BDNF contributes NDO maintenance. During spinal shock, BDNF sequestration resulted in early development of bladder hyperactivity, accompanied by increased axonal growth of calcitonin gene-related peptide-labeled fibers in the dorsal horn. Chronic BDNF administration inhibited the emergence of NDO, together with reduction of axonal growth, suggesting that BDNF may have a crucial role in bladder function after SCI via inhibition of neuronal sprouting. These findings highlight the role of BDNF in NDO and may provide a significant contribution to create more efficient therapies to manage SCI patients., (Copyright © 2015 Frias et al.)

The simultaneous delivery of multiple cancer drugs in combination therapies to achieve optimal therapeutic effects in patients can be challenging. This study investigated whether co-encapsulation of the BH3-mimetic ABT-737 and the topoisomerase I inhibitor camptothecin (CPT) in PEGylated polymeric nanoparticles (NPs) was a viable strategy for overcoming their clinical limitations and to deliver both compounds at optimal ratios. We found that thrombocytopenia induced by exposure to ABT-737 was diminished through its encapsulation in NPs. Similarly, CPT-associated leukopenia and gastrointestinal toxicity were reduced compared with the administration of free CPT. In addition to the reduction of dose-limiting side effects, the co-encapsulation of both anticancer compounds in a single NP produced synergistic induction of apoptosis in both in vitro and in vivo colorectal cancer models. This strategy may widen the therapeutic window of these and other drugs and may enhance the clinical efficacy of synergistic drug combinations.

The bladder mucosa consists of the urothelium, basement membrane, and lamina propria (LP). Although the urothelium has been given much attention, it may be regarded as one part of a signaling system involving another equally important component of the bladder mucosa, namely, the LP. The LP lies between the basement membrane of the mucosa and the detrusor muscle and is composed of an extracellular matrix containing several types of cells, including fibroblasts, adipocytes, interstitial cells, and afferent and efferent nerve endings. In addition, the LP contains a rich vascular network, lymphatic vessels, elastic fibers, and smooth muscle fascicles (muscularis mucosae). The roles of the LP and its components in bladder function have not been definitively established, though it has been suggested to be the capacitance layer of the bladder, determining bladder compliance and enabling adaptive changes to increasing volumes. However, the bladder LP may also serve as a communication center, with an important integrative role in signal transduction to the central nervous system (nociception, mechanosensation). The LP may also, by means of its different components, make it possible for the urothelium to transmit information to other components of the bladder wall, contributing to activation of the detrusor muscle. In addition, the LP may serve as a source for production of factors influencing the growth of both the overlying urothelium and the underlying detrusor muscle., (© 2013 Wiley Periodicals, Inc.)

Background and Purpose: The aim of the study was to determine whether KCNQ channels are functionally expressed in bladder smooth muscle cells (SMC) and to investigate their physiological significance in bladder contractility., Experimental Approach: KCNQ channels were examined at the genetic, protein, cellular and tissue level in guinea pig bladder smooth muscle using RT-PCR, immunofluorescence, patch-clamp electrophysiology, calcium imaging, detrusor strip myography, and a panel of KCNQ activators and inhibitors., Key Results: KCNQ subtypes 1-5 are expressed in bladder detrusor smooth muscle. Detrusor strips typically displayed TTX-insensitive myogenic spontaneous contractions that were increased in amplitude by the KCNQ channel inhibitors XE991, linopirdine or chromanol 293B. Contractility was inhibited by the KCNQ channel activators flupirtine or meclofenamic acid (MFA). The frequency of Ca²⁺-oscillations in SMC contained within bladder tissue sheets was increased by XE991. Outward currents in dispersed bladder SMC, recorded under conditions where BK and KATP currents were minimal, were significantly reduced by XE991, linopirdine, or chromanol, and enhanced by flupirtine or MFA. XE991 depolarized the cell membrane and could evoke transient depolarizations in quiescent cells. Flupirtine (20 μM) hyperpolarized the cell membrane with a simultaneous cessation of any spontaneous electrical activity., Conclusions and Implications: These novel findings reveal the role of KCNQ currents in the regulation of the resting membrane potential of detrusor SMC and their important physiological function in the control of spontaneous contractility in the guinea pig bladder., (© 2013 The Authors. British Journal of Pharmacology published by John Wiley &. Sons on behalf of The British Pharmacological Society.)

Objective: To characterize passive and active changes in detrusor activity in a highly compliant bladder., Materials and Methods: Bladders from adult female Sprague-Dawley rats were used 5 weeks after lower thoracic (T8) spinal cord transection or a sham-operation. Passive wall properties were assessed by pressure-volume relationships from whole bladders and the tensile response of bladder strips after a rapid (<0.5 s) stretch. Active properties were assessed from the frequency and amplitude of spontaneous contractions of bladder strips, and their response to the inotropic TRPV4 agonist GSK1016790A., Results: Passive bladder wall stiffness of SCT bladders was significantly reduced compared to that of the sham-operated control group (N = 6 and 8, respectively) and SCT bladder strips relaxed more quickly than those from sham-operated rats. The frequency of spontaneous contractions was reduced in SCT rats, and their amplitude, expressed as a ratio of bladder wall stiffness, was greater than in sham-operated rats. GSK1016790A (0.1 μM) significantly increased amplitude in strips from both sham-operated and SCT groups., Conclusions: There is no evidence of contractile failure in a highly-compliant bladder. The observations of reduced passive bladder wall stiffness and an enhanced rate of stress relaxation lead to the conclusion that increased compliance is marked by altered matrix properties that dissipate muscle force, thereby generating low pressures. Contractile agonists may be effective for improving bladder function in detrusor underactivity., (© 2012 BJU International.)

The field of bladder research has been energized by the study of novel interstitial cells (IC) over the last decade. Several subgroups of IC are located within the bladder wall and make structural interactions with nerves and smooth muscle, indicating integration with intercellular communication and key physiological functions. Significant progress has been made in the study of bladder ICs' cellular markers, ion channels and receptor expression, electrical and calcium signalling, yet their specific functions in normal bladder filling and emptying remain elusive. There is increasing evidence that the distribution of IC is altered in bladder pathophysiologies suggesting that changes in IC may be linked with the development of bladder dysfunction. This article summarizes the current state of the art of our knowledge of IC in normal bladder and reviews the literature on IC in dysfunctional bladder., (© 2012 The Author Acta Physiologica © 2012 Scandinavian Physiological Society.)

Several populations of interstitial cells of Cajal (ICC) exist in the bladder, associated with intramural nerves. Although ICC respond to exogenous agonists, there is currently no evidence of their functional innervation. The objective was to determine whether bladder ICC are functionally innervated. Guinea-pig bladder tissues, loaded with fluo-4AM were imaged with fluorescent microscopy and challenged with neurogenic electrical field stimulation (EFS). All subtypes of ICC and smooth muscle cells (SMC) displayed spontaneous Ca(2+)-oscillations. EFS (0.5 Hz, 2 Hz, 10 Hz) evoked tetrodotoxin (1 µM)-sensitive Ca(2+)-transients in lamina propria ICC (ICC-LP), detrusor ICC and perivascular ICC (PICC) associated with mucosal microvessels. EFS responses in ICC-LP were significantly reduced by atropine or suramin. SMC and vascular SMC (VSM) also responded to EFS. Spontaneous Ca(2+)-oscillations in individual ICC-LP within networks occurred asynchronously whereas EFS evoked coordinated Ca(2+)-transients in all ICC-LP within a field of view. Non-correlated Ca(2+)-oscillations in detrusor ICC and adjacent SMC pre-EFS, contrasted with simultaneous neurogenic Ca(2+) transients evoked by EFS. Spontaneous Ca(2+)-oscillations in PICC were little affected by EFS, whereas large Ca(2+)-transients were evoked in pre-EFS quiescent PICC. EFS also increased the frequency of VSM Ca(2+)-oscillations. In conclusion, ICC-LP, detrusor ICC and PICC are functionally innervated. Interestingly, Ca(2+)-activity within ICC-LP networks and between detrusor ICC and their adjacent SMC were synchronous under neural control. VSM and PICC Ca(2+)-activity was regulated by bladder nerves. These novel findings demonstrate functional neural control of bladder ICC. Similar studies should now be carried out on neurogenic bladder to elucidate the contribution of impaired nerve-ICC communication to bladder pathophysiology.

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., (Copyright © 2012 American Urological Association Education and Research, Inc. Published by Elsevier Inc. All rights reserved.)

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., (© 2011 The Authors Journal of Cellular and Molecular Medicine © 2011 Foundation for Cellular and Molecular Medicine/Blackwell Publishing Ltd.)

Purpose: We investigated the ultrastructural characteristics of interstitial cells of Cajal in the guinea pig bladder., Materials and Methods: Bladders were removed from guinea pigs and processed for transmission electron microscopy. Some sections were labeled with c-Kit antibodies and colloidal gold particles for positive identification of interstitial cells of Cajal., Results: Kit positive cells, identified with 10 nm gold particles, were located on the periphery of detrusor smooth muscle bundles and in the interbundle spaces. Interstitial cells of Cajal in these regions contained mitochondria, rough and smooth endoplasmic reticulum, thin and intermediate filaments, caveolae, Golgi apparatus, free ribosomes, cytoplasmic vesicles and had a discontinuous basal lamina. They were distinct from smooth muscle cells by an absence of dense bodies, membrane attachment bands and thick filaments. The ultrastructure of interstitial cells of Cajal in all regions of the bladder wall examined were similar and the myofibroblast characteristic, fibronexus, was not evident in any of the cells examined. Interstitial cells of Cajal had lateral branches which extended toward other interstitial cells of Cajal, neighboring smooth muscle cells or nerves. Cells with the ultrastructural profile of interstitial cells of Cajal were associated with bladder microvessels and their branched processes were in close proximity to vascular smooth muscle cells., Conclusions: Bladder interstitial cells of Cajal have typical ultrastructural characteristics of interstitial cells of Cajal from other tissues and were immunopositive for the interstitial cell of Cajal marker Kit. They were closely associated with detrusor smooth muscle and often networked with other interstitial cells of Cajal. The observation of perivascular cells with interstitial cells of Cajal characteristics indicates that there may be more subtypes of these cells in the bladder than previously considered., (Copyright © 2011 American Urological Association Education and Research, Inc. Published by Elsevier Inc. All rights reserved.)

Although members of the p63 family of transcription factors are known for their role in the development and differentiation of epithelial surfaces, their function in cancer is less clear. Here, we show that depletion of the ΔNp63α and β isoforms, leaving only ΔNp63γ, results in epithelial to mesenchymal transition (EMT) in the normal breast cell line MCF10A. EMT can be rescued by the expression of the ΔNp63α isoform. We also show that ΔNp63γ expressed in a background where all the other ΔNp63 are knocked down causes EMT with an increase in TGFβ-1, -2, and -3 and downstream effectors Smads2/3/4. In addition, a p63 binding site in intron 1 of TGFβ was identified. Inhibition of the TGFβ response with a specific inhibitor results in reversion of EMT in ΔNp63α- and β-depleted cells. In summary, we show that p63 is involved in inhibiting EMT and reduction of certain p63 isoforms may be important in the development of epithelial cancers.

The study of novel interstitial cells in the tissues of the urinary tract has defined advances in the field in the last decade. These intriguing cells belong to the same family as the better known interstitial cells of Cajal (ICC) of the gastrointestinal tract, and their discovery has been interpreted to suggest that pacemaker cells may be present in the urinary tract, driving the spontaneous or myogenic activity of the neighboring smooth muscle. This scenario may be true for the urethra where ICC have been described as "loose pacemakers" providing multiple, random inputs to modulate urethral smooth muscle activity. However, there is a paucity of direct evidence available to support this hypothesis in the bladder (where the smooth muscle cells are spontaneously active) or the renal pelvis (where atypical smooth muscle cells are the pacemakers), and it now seems more likely that urinary tract ICC act as modulators of smooth muscle activity.Interestingly, the literature suggests that the role of urinary tract ICC may be more apparent in pathophysiological conditions such as the overactive bladder. Several reports have indicated that the numbers of ICC present in overactive bladder tissues are greater than those from normal tissues; moreover, the contractility of tissues from overactive bladders in vitro appears to be more sensitive to the Kit antagonist, glivec, than those from normal bladder. Future research on urinary tract ICC in the short to medium term is likely to be dynamic and exciting and will lead to increasing our understanding of the roles of these cells in both normal and dysfunctional 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., (Copyright (c) 2010 American Urological Association Education and Research, Inc. Published by Elsevier Inc. All rights reserved.)

Aims: This review summarizes the currently available literature on the localization and proposed functions of a novel group of cells in the urinary bladder known as interstitial cells or interstitial cells of Cajal (ICC)., Methods: On-line searches of "Pubmed" for bladder, c-Kit, ICC, interstitial cell and myofibroblast were performed to identify relevant studies for the review., Results: The literature contains substantial data that several sub-populations of ICC are present in the wall of the mammalian urinary bladder. These are located in the lamina propria and within the detrusor with distinctive cell shapes and morphological arrangements. Bladder ICC are identified with transmission electron microscopy or by immunohistochemical labeling using antibodies to the Kit receptor which is an established ICC marker. Lamina propria-ICC form a loose network connected via Cx43 gap junctions and are associated with mucosal nerves. Detrusor ICC track the smooth muscle bundles and make frequent contacts with intramural nerves. Both groups of ICC exhibit spontaneous electrical and Ca2+-signalling and also respond to application of neurotransmitter substances including ATP and carbachol. There is emerging evidence that the expression of ICC is upregulated in pathophysiological conditions including the overactive bladder., Conclusions: There is now a convincing body of evidence that specialized ICC are present in the urinary bladder making important associations with other cells that make up the bladder wall and possessing physiological properties consistent with a role of bladder activity modulation.

Purpose: The presence of novel KCNQ currents was investigated in guinea pig bladder interstitial cells of Cajal and their contribution to the maintenance of the resting membrane potential was assessed., Materials and Methods: Enzymatically dispersed interstitial cells of Cajal were patch clamped with K(+) filled pipettes in voltage clamp and current clamp modes. Pharmacological modulators of KCNQ channels were tested on membrane currents and the resting membrane potential., Results: Cells were stepped from -60 to 40 mV to evoke voltage dependent currents using a modified K(+) pipette solution containing ethylene glycol tetraacetic acid (5 mM) and adenosine triphosphate (3 mM) to eliminate large conductance Ca activated K channel and K(adenosine triphosphate) currents. Application of the KCNQ blockers XE991, linopirdine (Tocris Bioscience, Ellisville, Missouri) and chromanol 293B (Sigma) decreased the outward current in concentration dependent fashion. The current-voltage relationship of XE991 sensitive current revealed a voltage dependent, outwardly rectifying current that activated positive to -60 mV and showed little inactivation. The KCNQ openers flupirtine and meclofenamic acid (Sigma) increased outward currents across the voltage range. In current clamp mode XE991 or chromanol 293B decreased interstitial cell of Cajal resting membrane potential and elicited the firing of spontaneous transient depolarizations in otherwise quiescent cells. Flupirtine or meclofenamic acid hyperpolarized interstitial cells of Cajal and inhibited any spontaneous electrical activity., Conclusions: This study provides electrophysiological evidence that bladder interstitial cells of Cajal have KCNQ currents with a role in the regulation of interstitial cell of Cajal resting membrane potential and excitability. These novel findings provide key information on the ion channels present in bladder interstitial cells of Cajal and they may indicate relevant targets for the development of new therapies for bladder instability.

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 Ca(2+) indicator fluo 4AM. ICC fired Ca(2+) transients in response to stimulation by carbachol (1/10 microM). Their pharmacology was consistent with carbachol-induced contractions in strips of detrusor which were inhibited by 4-DAMP (1 microM), an M(3) receptor antagonist, but not by the M(2) receptor antagonist methoctramine (1 microM). The source of Ca(2+) underlying the carbachol transients in isolated ICC was investigated using agents to interfere with influx or release from intracellular stores. Nifedipine (1 microM) or Ni(2+) (30-100 microM) to block Ca(2+) channels or the removal of external Ca(2+) reduced the amplitude of the carbachol transients. Application of ryanodine (30 microM) or tetracaine (100 microM) abolished the transients. The phospholipase C inhibitor, U-73122 (2.5 microM), significantly reduced the responses. 2-Aminoethoxydiethylborate (30 microM) caused a significant reduction and Xestospongin C (1 microM) was more effective, almost abolishing the responses. Intact in situ preparations of guinea pig bladder loaded with a Ca(2+) indicator showed distinctively different patterns of spontaneous Ca(2+) 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 Ca(2+) transients.

Objective: To identify interstitial cells (ICs) in the wall of the rabbit urethra using antibodies to the Kit receptor, and to examine their location, morphology and relationship with nerves and smooth muscle cells (SMCs), as studies of enzymatically isolated cells from the rabbit urethra have established that there are specialized cells that show spontaneous electrical activity and have morphological properties of ICs., Materials and Methods: Urethral tissues from rabbits were fixed, labelled with antibodies and examined with confocal microscopy. Some specimens were embedded in paraffin wax and processed for histology. Histological sections from the most proximal third and mid-third region of rabbit urethra were stained with Masson's Trichrome to show their cellular arrangement., Results: Sections from both regions had outer longitudinal and inner circular layers of SM, and a lamina propria containing connective tissue and blood vessels; the lumen was lined with urothelial cells. The mid-third region had a more developed circular SM layer than the most-proximal samples, and had extensive inner longitudinal SM bundles in the lamina propria. Labelling with anti-Kit revealed immunopositive cells within the wall of the rabbit urethra, in the circular and longitudinal layers of the muscularis. Double-labelling with an antibody to SM myosin showed Kit-positive cells on the boundary of the SM bundles, orientated parallel to the axis of the bundles. Others were in spaces between the bundles and often made contact with each other. Kit-positive cells were either elongated, with several lateral branches, or stellate with branches coming from a central soma. Similar cells could be labelled with vimentin antibodies. Their relationship with intramural nerves was examined by double immunostaining with an anti-neurofilament antibody. There were frequent points of contact between Kit-positive cells and nerves, with similar findings in specimens double-immunostained with anti-neuronal nitric oxide synthase (nNOS)., Conclusion: Kit-positive ICs were found within the SM layers of the rabbit urethra, in association with nerves, on the edge of SM bundles and in the interbundle spaces. The contact with nNOS-containing neurones might imply participation in the nitrergic inhibitory neurotransmission of the urethra.

Objective: To determine whether there are inward currents in interstitial cells (IC) isolated from the guinea-pig detrusor and if so, to characterise them using the patch-clamp technique and pharmacological agents., Materials and Methods: Using the whole-cell patch-clamp technique, inward currents were studied in IC enzymatically isolated from the detrusor of the guinea-pig bladder. Currents were evoked by stepping positively from a holding potential of - 80 mV., Results: Outward K+ currents were blocked by Cs+ internal solution to reveal inward currents, which activated at voltages more positive than - 50 mV, peaked at 0 mV, reversed near + 50 mV and were half-maximally activated at - 27 mV. The inward currents showed voltage-dependent inactivation and were half-maximally inactivated at - 36 mV. Fitting the activation and inactivation data with a Boltzmann function revealed a window current between - 40 mV and + 20 mV. The decay of the current evoked at 0 mV could be fitted with a single exponential with a mean time-constant of 88 ms. Replacing external Ca2+ with Ba2+ significantly increased this to 344 ms. The current amplitude was augmented by Ba2+, and by Bay K 8644. Inward currents were significantly reduced by 1 microm nifedipine, across the voltage range, but the blockade was more effective on the current evoked at 0 mV than that evoked by a step to - 20 mV, perhaps indicating voltage-dependence of the action of nifedipine or another component of inward current. Increasing the concentration of the drug to 10 microm caused no further significant reduction either at 0 mV or at -20 mV. However, in the presence of 1 microm nifedipine the latter current was significantly reduced by 100 microm Ni2+. Both currents were significantly reduced in Ca2+-free solution., Conclusions: IC from the guinea-pig detrusor possess inward currents with typical characteristics of L-type Ca2+ current. They also have a component of inward Ca2+ current, which was resistant to nifedipine, but sensitive to Ni2+. Further work is needed to characterise the latter conductance.

Scientists interested in the smooth muscles of the urinary tract, and their control, have recently been studying cells in the interstitium of tissues that express the c-kit antigen (Kit(+) cells). These cells have morphologic features that are reminiscent of the well-described pacemaker cells in the gut, the interstitial cells of Cajal (ICC). The spontaneous contractile behavior of muscles in the urinary tract varies widely, and it is clear that urinary tract Kit(+) interstitial cells cannot be playing an identical role to that played by the ICC in the gut. Nevertheless, there is increasing evidence that they do play a role in modulating the contractile behavior of adjacent smooth muscle, and might also be involved in mediating neural control. This review outlines the properties of ICC in the gut, and gives an account of the discovery of cells in the interstitium of the main components of the urinary tract. The physiologic properties of such cells and the functional implications of their presence are discussed, with particular reference to the bladder. In this organ, Kit(+) cells are found under the lamina propria, where they might interact with the urothelium and with sensory nerves, and also between and within the smooth-muscle bundles. Confocal microscopy and calcium imaging are being used to assess the physiology of ICC and their interactions with smooth muscles. Differences in the numbers of ICC are seen in smooth muscle specimens obtained from patients with various pathologies; in particular, bladder overactivity is associated with increased numbers of these cells.

Purpose: In the current study we examined the location of interstitial cell of Cajal (ICC)-like cells in the guinea pig bladder wall and studied their structural interactions with nerves and smooth muscle cells., Materials and Methods: Whole mount samples and cryosections of bladder tissue were labeled with primary and fluorescent secondary antibodies, and imaged using confocal and multiphoton microscopy., Results: Kit positive ICC-like cells were located below the urothelium, in the lamina propria region and throughout the detrusor. In the suburothelium they had a stellate morphology and appeared to network. They made connections with nerves, as shown by double labeling experiments with anti-kit and anti-protein gene product 9.5. A network of vimentin positive cells was also found, of which many but not all were kit positive. In the detrusor kit positive cells were most often seen at the edge of smooth muscle bundles. They were elongated with lateral branches, running in parallel with the bundles and closely associated with intramural nerves. Another population of kit positive cells was seen in the detrusor between muscle bundles. These cells had a more stellate-like morphology and made connections with each other. Kit positive cells were seen tracking nerve bundles and close to intramural ganglia. Vimentin positive cells were present in the detrusor, of which some were also kit positive., Conclusions: There are several populations of ICC-like cells throughout the guinea pig bladder wall. They differ in morphology and orientation but all make connections with intramural nerves and in the detrusor they are closely associated with smooth muscle cells.

Purpose: Outward currents were characterized from cells resembling interstitial cells of Cajal (ICCs) isolated from the detrusor of the guinea pig bladder., Materials and Methods: ICC-like cells were studied using the whole cell patch clamp technique and K+ filled pipettes. Outward currents were evoked by stepping positively from a holding potential of -80 mV., Results: ICC-like cells were distinguished from smooth muscle cells by the presence of lateral branches and an inability to contract spontaneously or when depolarized. Depolarization elicited large outward currents. Penitrem A, a blocker of large conductance, Ca activated K+ channels, significantly decreased the outward current. Its Ca dependence was demonstrated by significant inhibition with nifedipine and Ca-free solution. When large conductance, Ca activated K+ and Ca currents were blocked with penitrem A and nifedipine, a voltage dependent current was unmasked, which activated positive to -50 mV and displayed voltage dependent inactivation with half-maximal inactivation occurring at -71 mV. It was blocked in concentration dependent fashion by tetraethylammonium but unaffected by 4-aminopyridine, charybdotoxin or apamin, suggesting that small and intermediate conductance, calcium activated potassium channels, and Kv1.2 and Kv1.3 channels are unlikely to be involved. At maximal concentrations of tetraethylammonium a portion of the voltage dependent K+ current remained that was not affected by any of the blockers tested., Conclusions: ICC-like cells from the detrusor possess calcium activated and voltage dependent K+ currents.

Recent electrophysiological studies have suggested that there is a subpopulation of cells in lymphatic vessels which act as pacemakers controlling the characteristic spontaneous contractile activity in this tissue. In this study, electron microscopy and immunohistochemical techniques were used on sheep mesenteric lymphatic vessels to investigate the morphology of the cells comprising the lymphatic wall. The smooth muscle cells were not orientated in circular and longitudinal layers as is seen in the gastrointestinal tract, but were arranged in bundles which interlock and cross over in a basket-weave fashion. Antibodies to Kit and vimentin, which are widely used to label specialised pacemaking cells in the gastrointestinal tract (known as interstitial cells of Cajal), demonstrated the existence of an axially orientated subpopulation of cells lying between the endothelium and the bulk of the smooth muscle. Examination of this area using electron microscopy showed cells which were electron dense compared to the underlying smooth muscle and contained caveolae, Golgi complexes, mitochondria, 10-nm filaments, a well-developed endoplasmic reticulum and a basal lamina. The smooth muscle cells typically contained caveolae, dense bodies, mitochondria, abundant filaments, sER and basal laminae. Cells dispersed for patch-clamp studies were also stained for vimentin and myosin. Myosin-staining cells had the typical spindle appearance of smooth muscle cells whereas the vimentin-positive cells could either be branched or more closely resemble the smooth muscle cells. The present study provides the first morphological evidence that specialised cells exist within the vascular system which have the ultrastructural characteristics of pacemaker cells in other tissues and are vimentin and Kit positive.

Purpose: We describe the presence of interstitial cells of Cajal (ICC) throughout the wall of the guinea pig bladder., Materials and Methods: Bladders obtained from male guinea pigs were prepared for immunohistochemical investigations using various primary antibodies, including the specific ICC marker c-kit (Gibco BRL, Grand Island, New York). Enzymatically dispersed cells with a branched morphology were identified as ICC using anti-c-kit. They were loaded with fluo-4acetoxymethyl (Molecular Probes, Eugene, Oregon) and studied using confocal laser scanning microscopy., Results: Anti-c-kit labeling demonstrated that ICC were oriented in parallel with the smooth muscle bundles that run diagonally throughout the bladder. Double labeling with anti-smooth muscle myosin (Sigma Chemical Co., St. Louis, Missouri) revealed that ICC were located on the boundary of smooth muscle bundles. When anti-c-kit was used in combination with the general neuronal antibody protein gene product 9.5 (Ultraclone Ltd., Isle of Wight, United Kingdom) or anti-neuronal nitric oxide synthase, it was noted that there was a close association between nerves and ICC. Enzymatic dissociation of cells from tissue pieces yielded a heterogeneous population of cells containing typical spindle-shaped smooth muscle cells and branched cells resembling ICC from other preparations. The latter could be identified immunohistochemically as ICC using anti-c-kit, whereas the majority of spindle-shaped cells were not Kit positive. Branched cells responded to the application of carbachol by firing Ca2+ waves and they were often spontaneously active., Conclusions: ICC are located on the boundary of smooth muscle bundles in the guinea pig bladder. They fire Ca2+ waves in response to cholinergic stimulation and can be spontaneously active, suggesting that they could act as pacemakers or intermediaries in the transmission of nerve signals to smooth muscle cells.

Isolated interstitial ("pacemaker") cells from rabbit urethra were examined using the perforated-patch technique. Under voltage clamp at -60 mV, these cells fired large spontaneous transient inward currents (STICs), averaging -860 pA and >1 s in duration, which could account for urethral pacemaker activity. Spontaneous transient outward currents (STOCs) were also observed and fell into two categories, "fast" (<100 ms in duration) and "slow" (>1 s in duration). The latter were coupled to STICs, suggesting that they shared the same mechanism, while the former occurred independently at faster rates. All of these currents were abolished by cyclopiazonic acid, caffeine, or ryanodine, suggesting that they were activated by Ca(2+) release. When D-myo-inositol 1,4,5-trisphosphate (IP(3))-sensitive stores were blocked with 2-aminoethoxydiphenyl borate, the STICs and slow STOCs were abolished, but the fast STOCs remained. In contrast, the fast STOCs were more nifedipine sensitive than the STICs or the slow STOCs. These results suggest that while fast STOCs are mediated by a mechanism similar to STOCs in smooth muscle, STICs and slow STOCs are driven by IP(3). These results support the hypothesis that pacemaker activity in the urethra is driven by the IP(3)-sensitive store.

Freshly dispersed sheep mesenteric lymphatic smooth muscle cells were studied at 37 degrees C using the perforated patch-clamp technique with Cs(+)- and K(+)-filled pipettes. Depolarizing steps evoked currents that consisted of L-type Ca(2+) [I(Ca(L))] current and a slowly developing current. The slow current reversed at 1 +/- 1.5 mV with symmetrical Cl(-) concentrations compared with 23.2 +/- 1.2 mV (n = 5) and -34.3 +/- 3.5 mV (n = 4) when external Cl(-) was substituted with either glutamate (86 mM) or I(-) (125 mM). Nifedipine (1 microM) blocked and BAY K 8644 enhanced I(Ca(L)), the slow-developing sustained current, and the tail current. The Cl(-) channel blocker anthracene-9-carboxylic acid (9-AC) reduced only the slowly developing inward and tail currents. Application of caffeine (10 mM) to voltage-clamped cells evoked currents that reversed close to the Cl(-) equilibrium potential and were sensitive to 9-AC. Small spontaneous transient depolarizations and larger action potentials were observed in current clamp, and these were blocked by 9-AC. Evoked action potentials were triphasic and had a prominent plateau phase that was selectively blocked by 9-AC. Similarly, fluid output was reduced by 9-AC in doubly cannulated segments of spontaneously pumping sheep lymphatics, suggesting that the Ca(2+)-activated Cl(-) current plays an important role in the electrical activity underlying spontaneous activity in this tissue.

The perforated-patch technique was used to measure membrane currents in smooth muscle cells from sheep urethra. Depolarizing pulses evoked large transient outward currents and several components of sustained current. The transient current and a component of sustained current were blocked by iberiotoxin, penitrem A, and nifedipine but were unaffected by apamin or 4-aminopyridine, suggesting that they were mediated by large-conductance Ca(2+)-activated K(+) (BK) channels. When the BK current was blocked by exposure to penitrem A (100 nM) and Ca(2+)-free bath solution, there remained a voltage-sensitive K(+) current that was moderately sensitive to blockade with tetraethylammonium (TEA; half-maximal effective dose = 3.0 +/- 0.8 mM) but not 4-aminopyridine. Penitrem A (100 nM) increased the spike amplitude and plateau potential in slow waves evoked in single cells, whereas addition of TEA (10 mM) further increased the plateau potential and duration. In conclusion, both Ca(2+)-activated and voltage-dependent K(+) currents were found in urethral myocytes. Both of these currents are capable of contributing to the slow wave in these cells, suggesting that they are likely to influence urethral tone under certain conditions.