Christopher H. Fry

T-type Ca²⁺ channels are widely expressed throughout the urinary and male genital tracts, generally alongside L-type Ca²⁺ channels. The use of pharmacological blockers of these channels has suggested functional roles in all regions, with the possible exception of the ureter. Their functional expression is apparent not just in smooth muscle cells but also in interstitial cells that lie in close proximity to muscle, nerve and epithelial components of these tissues. Thus, T-type Ca²⁺ channels can contribute directly to modulation of muscle function and indirectly to changes of epithelial and nerve function. T-type Ca²⁺ channel activity modulates phasic contractile activity, especially in conjunction with Ca²⁺-activated K⁺ channels, and also to agonist-dependent responses in different tissues. Upregulation of channel density occurs in pathological conditions associated with enhanced contractile responses, e.g. overactive bladder, but it is unclear if this is causal or a response to the pathological state. Moreover, T-type Ca²⁺ channels may have a role in the development of prostate tumours regulating the secretion of mitogens from neuroendocrine cells. Although a number of selective channel blockers exist, their relative selectivity over L-type Ca²⁺ channels is often low and makes evaluation of T-type Ca²⁺ channel function in the whole organism difficult.

Aims A biomarker is an entity that measures a normal or pathological process, or the response to an intervention. A biomarker must measure exclusively and be sufficiently sensitive to the process of interest. Alternatively, a biomarker may give clues regarding the underlying pathology of the condition and be a useful research or specialist tool. If a biomarker is to be of practical benefit then it must also be economical and practical to use. This article will consider chemical moieties as biomarkers, although in principle physical markers (e.g., bladder wall thickness) could also be defined as such. Results and Conclusions The validation of a biomarker for detrusor overactivity (DO) must appreciate the fact that the condition is likely to multifactorial and thus no single entity may be sufficiently selective and sensitive. However, more specific conditions, such as bladder pain associated with DO, may make the biomarker search easier. Several prospective agents including antiproliferative factor (APF) and epidermal growth factors (EGF) are discussed. Several urinary biomarkers, including neurotrophins (NGF, BDNF) and cytokines, and a serum marker, C-reactive protein, are considered as reaching the above criteria. All suffer from relatively poor lack of discrimination, as they all change in response to other, often inflammatory, conditions; BDNF may offer the highest expectations. Urinary ATP has also been proposed as a DO/OAB biomarker but requires further evaluation. Finally genetic markers offer potential to understand more about the pathophysiology of DO/OAB. The increasing availability of genome-wide association studies and micro-RNA assays offer genetic markers as a new generation of biomarkers. Neurourol. Urodynam. 33:602–605, 2014.

What's known on the subject? and What does the study add?: Stretch of the urothelium, as occurs during bladder filling, is associated with a release of ATP that is postulated to act as a sensory neurotransmitter. The regulation of ATP release is poorly understood and in particular if there is a feedback mechanism provided by ATP itself. Adenosine, a breakdown product of ATP , is a potent inhibitor of stretch‐induced ATP release, acting through and A1 receptor; endogenous levels are about 0.6μM. Data are consistent with ATP release relying on the rise of intracellular Ca2+. Transepithelial potential also controls ATP release, also acting via an A1 receptor‐dependent pathway. Objectives: To test the hypothesis that distension‐induced ATP release from the bladder urothelium is regulated by adenosine as well as changes to transurothelial potential ( TEP ). To examine the role of changes to intracellular [Ca ²⁺ ] in ATP release. Materials and Methods: Rabbit urothelium/suburothelium membranes were used in an Ussing chamber system. Distension was induced by fluid removal from the chamber bathing the serosal (basolateral) membrane face. The TEP and short‐circuit current were measured. ATP was measured in samples aspirated from the serosal chamber by a luciferin–luciferase assay. Intracellular [Ca ²⁺ ] was measured in isolated urothelial cells using the fluorochrome Fura‐2. All experiments were performed at 37°C. Results: Distension‐induced ATP release was decreased by adenosine (1–10 μ m ) and enhanced by adenosine deaminase and A1‐ (but not A2‐) receptor antagonists. Distension‐induced ATP release was reduced by 2‐APB, nifedipine and capsazepine; capsaicin induced ATP release in the absence of distension. ATP and capsaicin, but not adenosine, generated intracellular Ca ²⁺ transients; adenosine did not affect the ATP ‐generated Ca ²⁺ transient. ATP release was dependent on a finite transepithelial potential. Changes to TEP , in the absence of distension, generated ATP release that was in turn reduced by adenosine. Conclusion: Adenosine exerts a powerful negative feedback control of ATP release from the urothelium via A1 receptor activation. Distension‐induced ATP release may be mediated by a rise of the intracellular [Ca ²⁺ ]. Modulation of distension‐induced ATP release by adenosine and TEP may have a common pathway.

What's known on the subject? and What does the study add?: 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. Objective: To characterize passive and active changes in detrusor activity in a highly compliant bladder. Materials and Methods: Bladders from adult female S prague– D awley rats were used 5 weeks after lower thoracic ( T 8) 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 (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. GSK 1016790A (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.

Spinal cord transection (SCT) leads to an increase in spontaneous contractile activity in the isolated bladder that is reminiscent of an overactive bladder syndrome in patients with similar damage to the central nervous system. An increase in interstitial cell number in the suburothelial space between the urothelium and detrusor smooth muscle layer occurs in SCT bladders, and these cells elicit excitatory responses to purines and pyrimidines such as ATP, ADP, and UTP. We have investigated the hypothesis that these agents underlie the increase in spontaneous activity. Rats underwent lower thoracic spinal cord transection, and their bladder sheets or strips, with intact mucosa except where specified, were used for experiments. Isometric tension was recorded and propagating Ca²⁺and membrane potential ( E_m) waves were recorded by fluorescence imaging using photodiode arrays. SCT bladders were associated with regular spontaneous contractions (2.9 ± 0.4/min); ADP, UTP, and UDP augmented the amplitude but not their frequency. With strips from such bladders, a P2Y₆-selective agonist (PSB0474) exerted similar effects. Fluorescence imaging of bladder sheets showed that ADP or UTP increased the conduction velocity of Ca²⁺/ E_mwaves that were confined to regions of the bladder wall with an intact mucosa. When transverse bladder sections were used, Ca²⁺/ E_mwaves originated in the suburothelial space and propagated to the detrusor and urothelium. Analysis of wave propagation showed that the suburothelial space exhibited properties of an electrical syncitium. These experiments are consistent with the hypothesis that P2Y-receptor agonists increase spontaneous contractile activity by augmenting functional activity of the cellular syncitium in the suburothelial space.

What's known on the subject? and What does the study add? ATP and acetylcholine (ACh) are released from the urothelium or from cultured urothelial cells in response to mechanical stretching or perturbations that mimic stretching, such as exposure to hypotonic solutions. Limited evidence suggests that ATP and ACh may interact, as the muscarinic agonist carbachol evokes ATP release from cultured urothelial cells. Our aim was to determine whether antimuscarinics, shown previously to reduce sensory nerve firing during bladder filling, might function by reducing mucosal ATP release. By stretching mucosa strips and measuring superfusate ATP using a luciferin–luciferase assay, we showed that muscarinic receptor antagonism (4‐DAMP, 10 nM or methoctramine, 1 µM) inhibits stretching‐evoked ATP release, suggesting anticholinergic agents used to treat human lower urinary tract pathologies act on urothelial muscarinic receptors. We also showed that alteration of resting mucosal tension is the key determinant of ATP release, as ATP is released from the mucosa in response to relaxation, as well to stretching. OBJECTIVE: To determine whether muscarinic receptor antagonism affects stretching‐induced release of ATP. MATERIALS AND METHODS: Mucosal strips, dissected from guinea pig (male, 450g; n = 10) urinary bladders, were placed in horizontal organ baths and superfused with Ca ²⁺ ‐free Tyrode's solution. Superfusate samples were taken pre‐ and post‐ intervention (rapid stretching or relaxation) and ATP concentration was quantified using a luciferin–luciferase assay. The effect of muscarinic acetylcholine receptor antagonism on ATP release was assessed by addition of methoctramine (1 µM) and 4‐DAMP (10 nM). RESULTS: Rapid stretching (0 to 13.3 ± 1.2 mN; no. strips = 20) increased ATP in the superfusate to a median threefold increase over basal levels. After a period of equilibration, tension in the mucosal strips relaxed until it had reached a new steady‐state after 60 min and stretching was repeated. In the presence of 4‐DAMP (10 nM) or methoctramine (1 µM), ATP concentrations after stretching reduced to 61% or 20%, respectively. By contrast, ATP concentrations in mucosa‐matched controls, perfused with vehicle, increased in response to stretching by 391% and 1500%, respectively. Rapid relaxation also stimulated ATP release. This release did not appear to be sensitive to 4‐DAMP or methoctramine. CONCLUSIONS: An alteration of resting mucosal tension is the key determinant of ATP release, as ATP is released from the mucosa in response to both stretching and relaxation. Muscarinic receptor antagonism inhibits stretching‐evoked ATP release from bladder mucosa, suggesting that anticholinergic agents used to treat human lower urinary tract pathologies act on urothelial muscarinic receptors.

Models of the lower urinary tract are used to understand better the physiological and pathological functions of the tract and to gain insight into the relative importance of different components. The key requirement of a model is described, namely: to involve a continuous iteration with experiment; whereby experiments provide parameters and validation for components of the model, which is then used to generate hypotheses, which are tested experimentally. Different types of models are described: computational models that describe mathematically the whole urinary tract or components; physical models useful especially in testing medical devices; and tissue‐engineered models. The purpose of modeling is first described in terms of the ability of models to predict the properties of the system of interest, using components that have a physiological interpretation, and to gain insight into the relative importance of different components. Examples are used to illustrate the use of modeling the urinary tract with reference to the different categories listed above. Neurourol. Urodynam. Neurourol. Urodynam. 30:692–699, 2011.

What’s known on the subject? and What does the study add? The question to be addressed was whether bladder overactivity or obstruction in patients was associated with any changes to the contractile properties of the detrusor smooth muscle, and in addition were there age‐related and gender‐dependent differences in these? The study showed that neither age nor gender per se affected the contractile properties of the smooth muscle from the stable bladder. However, in the pathology groups there was an age‐dependent reduction of nerve‐dependent contractile function, and thus in the overactive bladder there is an age‐dependent functional deneravation. OBJECTIVE: • To test the hypothesis that the in vitro contractile properties of human detrusor smooth muscle are dependent on the age, gender and lower urinary tract pathology of the patient. MATERIALS AND METHODS: • Contractions were elicited in isolated human detrusor smooth muscle preparations by nerve‐mediated electrical field stimulation, agonist application (carbachol, α,β‐methylene ATP and high‐K solutions) or direct muscle electrical stimulation. • Biopsies ( n = 227) were obtained from four groups of patients with: stable bladders (control), bladder outlet obstruction (BOO), idiopathic (IDO), or neurogenic (NDO) detrusor overactivity. RESULTS: • The magnitude of nerve‐mediated contractions declined as a function of patients’ age in each of the BOO, IDO and NDO groups but not in the control group. • Contractions elicited by direct muscle activation (10 µ m carbachol or electrical stimulation with 20 ms pulses in the presence of 1 µ m tetrodotoxin) did not vary with patient age. • Carbachol contractions were significantly smaller in samples from NDO bladders. • Atropine resistance was more prevalent in the pathology groups compared with the control group and was greatest in the IDO group. There was no influence of age in the prevalence or magnitude of atropine‐resistant contractions in any group. • Muscle excitability to direct electrical stimulation was similar in all groups. CONCLUSIONS: • In the human bladder there is no evidence for a decline of detrusor smooth muscle contractility or excitability as a function of age, nor any gender difference or presence of pathology. • In the pathology groups there was evidence for a decline of functional innervation with age.