Kylie J Mansfield

Muscarinic receptor subtypes in human bladder detrusor and mucosa, studied by radioligand binding and quantitative competitive RT-PCR: changes in ageing.

1 We investigated muscarinic receptors in the detrusor and mucosa of the human bladder body. Radioligand‐binding studies with [3H]QNB were conducted using specimens collected from patients (36–77 years) with normal bladder function, undergoing surgery. For RT–PCR, biopsies of normal bladder were obtained from patients (30–88 years) undergoing check cystoscopy. 2 Binding of [3H]QNB in detrusor (n=20) was of high affinity (KD 77.1 (55.2–99.0) pM) and capacity (Bmax 181±7 fmol mg protein−1). Similar values were obtained in mucosa (n=6) (KD 100.5 (41.2–159.9) pM; Bmax 145±9 fmol mg protein−1). 3 Competition‐binding experiments in detrusor membranes with muscarinic receptor antagonists including trospium, darifenacin, 4‐DAMP, methoctramine, AQ‐RA 741, AF‐DX 116 and pirenzepine indicated a receptor population of 71% M2, 22% M3 and 7% M1. In the mucosa, 75% of sites were M2 receptors, with 25% M3/M5. 4 Using RT–PCR, expression of M1, M2, M3 and M5 mRNA was demonstrated in both detrusor and mucosa. 5 The presence of a high density of mainly M2 muscarinic receptors in the mucosa appears to be a novel finding and raises the question of their physiological significance and the source of their endogenous ligand. 6 There was a negative correlation of receptor number (Bmax) with age in detrusor muscle from male patients (P=0.02). Quantitative competitive RT–PCR demonstrated a selective age‐related decrease in mRNA for muscarinic M3 but not M2 receptors, in both male (P<0.0001) and female (P=0.019) detrusor. These findings correspond with reports of decreased detrusor contractility with ageing.

Host–pathogen checkpoints and population bottlenecks in persistent and intracellular uropathogenic Escherichia coli bladder infection

Bladder infections affect millions of people yearly, and recurrent symptomatic infections (cystitis) are very common. The rapid increase in infections caused by multidrug-resistant uropathogens threatens to make recurrent cystitis an increasingly troubling public health concern. Uropathogenic Escherichia coli (UPEC) cause the vast majority of bladder infections. Upon entry into the lower urinary tract, UPEC face obstacles to colonization that constitute population bottlenecks, reducing diversity, and selecting for fit clones. A critical mucosal barrier to bladder infection is the epithelium (urothelium). UPEC bypass this barrier when they invade urothelial cells and form intracellular bacterial communities (IBCs), a process which requires type 1 pili. IBCs are transient in nature, occurring primarily during acute infection. Chronic bladder infection is common and can be either latent, in the form of the quiescent intracellular reservoir (QIR), or active, in the form of asymptomatic bacteriuria (ASB/ABU) or chronic cystitis. In mice, the fate of bladder infection, QIR, ASB, or chronic cystitis, is determined within the first 24 h of infection and constitutes a putative host-pathogen mucosal checkpoint that contributes to susceptibility to recurrent cystitis. Knowledge of these checkpoints and bottlenecks is critical for our understanding of bladder infection and efforts to devise novel therapeutic strategies.

Release of ATP from rat urinary bladder mucosa: role of acid, vanilloids and stretch

Background and purpose:  ATP, released from urothelial cells, modulates afferent nerve firing from the urinary bladder. Here, we have characterized ATP release from the rat bladder mucosa in response to acid, capsaicin, electrical field stimulation (EFS) and stretch, using agonists and antagonists at transient receptor potential vanilloid receptor 1 (TRPV1) and acid‐sensing ion channels (ASICs).

Differential blockade of neuronal voltage-gated Na+ and K+ channels by antidepressant drugs

The effects of a range of antidepressants were investigated on neuronal voltage-gated Na(+) and K(+) channels. With the exception of phenelzine, all antidepressants inhibited batrachotoxin-stimulated 22Na(+) uptake, most likely via negative allosteric inhibition of batrachotoxin binding to neurotoxin receptor site-2 on the Na(+) channel. Imipramine also produced a differential action on macroscopic Na(+) and K(+) channel currents in acutely dissociated rat dorsal root ganglion neurons. Imipramine produced a use-dependent block of Na(+) channels. In addition, there was a hyperpolarizing shift in the voltage-dependence of steady-state Na(+) channel inactivation and slowed repriming kinetics consistent with imipramine having a higher affinity for the inactivated state of the Na(+) channel. At higher concentrations, imipramine also blocked delayed-rectifier and transient outward K(+) currents in the absence of alterations to the voltage-dependence of activation or the kinetics of inactivation. These actions on voltage-gated ion channels may underlie the therapeutic and toxic effects of these drugs.

Porcine bladder urothelial, myofibroblast, and detrusor muscle cells: characterization and ATP release

ATP is released from the bladder mucosa in response to stretch, but the cell types responsible are unclear. Our aim was to isolate and characterize individual populations of urothelial, myofibroblast, and detrusor muscle cells in culture, and to examine agonist-stimulated ATP release. Using female pig bladders, urothelial cells were isolated from bladder mucosa following trypsin-digestion of the luminal surface. The underlying myofibroblast layer was dissected, minced, digested, and cultured until confluent (10–14 days). A similar protocol was used for muscle cells. Cultures were used for immunocytochemical staining and/or ATP release investigations. In urothelial cultures, immunoreactivity was present for the cytokeratin marker AE1/AE3 but not the contractile protein α-smooth muscle actin (α-SMA) or the cytoskeletal filament vimentin. Neither myofibroblast nor muscle cell cultures stained for AE1/AE3. Myofibroblast cultures partially stained for α-SMA, whereas muscle cultures were 100% stained. Both myofibroblast and muscle stained for vimentin, however, they were morphologically distinct. Ultrastructural studies verified that the suburothelial layer of pig bladder contained abundant myofibroblasts, characterized by high densities of rough endoplasmic reticulum. Baseline ATP release was higher in urothelial and myofibroblast cultures, compared with muscle. ATP release was significantly stimulated by stretch in all three cell populations. Only urothelial cells released ATP in response to acid, and only muscle cells were stimulated by capsaicin. Tachykinins had no effect on ATP release. In conclusion, we have established a method for culture of three cell populations from porcine bladder, a well-known human bladder model, and shown that these are distinct morphologically, immunologically, and pharmacologically.

Comparison of Receptor Binding Characteristics of Commonly Used Muscarinic Antagonists in Human Bladder Detrusor and Mucosa

Recent studies have described muscarinic receptors on the mucosa and the detrusor of the human urinary bladder. Muscarinic receptor antagonists are effective in the treatment of overactive bladder (OAB), but their site(s) of action and actual therapeutic target are unclear. Our aim was to compare, in human bladder mucosa and detrusor, the radioligand binding characteristics of newer, clinically effective agents: darifenacin, its hydroxylated metabolite UK-148,993, fesoterodine, solifenacin, tolterodine, and trospium. Specimens were collected from asymptomatic patients (50-72 years old) undergoing open bladder surgery. Radioligand binding studies with the muscarinic antagonist [3H]quinuclidinyl benzilate (QNB) were performed separately on detrusor and mucosal membranes. All antagonists displayed high affinity when competing for [3H]QNB binding in both detrusor and mucosa. Inhibition constants were also obtained for all antagonists against individual muscarinic receptor subtypes expressed in Chinese hamster ovary cells. Here, fesoterodine showed anomalous binding results, suggesting that some conversion to its metabolite had occurred. Global nonlinear regression analysis of bladder binding data with five antagonists demonstrated 82% low-affinity sites in mucosa and 78% low-affinity sites in detrusor, probably representing M2/M4 receptors. There was an excellent correlation (r2 = 0.99) of low-affinity global estimates between detrusor and mucosa, whereas the corresponding high-affinity estimates (∼20% of sites) were dissimilar. In conclusion, commonly used and clinically effective muscarinic receptor antagonists bind to receptors located on the bladder mucosa and the detrusor, providing support for the hypothesis that muscarinic receptors in the mucosa may represent an important site of action for these agents in OAB.

Does Adenosine Triphosphate Released Into Voided Urodynamic Fluid Contribute to Urgency Signaling in Women With Bladder Dysfunction

PURPOSE Adenosine triphosphate released from urothelium during stretch stimulates afferent nerves and conveys information on bladder fullness. We measured adenosine triphosphate released during cystometric bladder filling in women with idiopathic detrusor overactivity and stress incontinence (controls), and assessed whether the level of released adenosine triphosphate is related to cystometric parameters. MATERIALS AND METHODS Routine cystometry was done in 51 controls and 48 women with detrusor overactivity who were 28 to 87 years old. Voided urodynamic fluid was collected and stored at -30 C. Adenosine triphosphate was measured by a bioluminescence assay. RESULTS Adenosine triphosphate levels were similar in voided urodynamic fluid of controls and patients with detrusor overactivity (p = 0.79). A significant inverse correlation was seen between adenosine triphosphate and maximal cystometric capacity in controls (p = 0.013), and between voided volume and adenosine triphosphate in controls (p = 0.015) and detrusor overactivity cases (p = 0.019). A significant correlation between first desire to void and adenosine triphosphate was also noted in detrusor overactivity cases (p = 0.033) but not in controls (p = 0.58). No correlation was seen between adenosine triphosphate and detrusor pressure during filling or voiding. CONCLUSIONS Adenosine triphosphate measurement in voided urodynamic fluid is a novel approach to understanding signals that may contribute to the urgency sensation (a sudden compelling desire to pass urine). The inverse correlation between adenosine triphosphate in voided urodynamic fluid and first desire to void suggests that adenosine triphosphate has a role in modulating the early filling sensation in patients with detrusor overactivity.

Molecular characterization of M2 and M3 muscarinic receptor expression in bladder from women with refractory idiopathic detrusor overactivity

In a study from Sydney, authors present data on the expression of M2 and M3 receptors in human detrusor and bladder mucosa, comparing patients with idiopathic detrusor overactivity with controls. It was found that M3 muscarinic receptor RNA expression was reduced in mucosa from patients with detrusor overactivity, leading to a proposal that mucosal M3 receptors might have an important role in the aetiology of this condition.

A Cross-Sectional Comparison of Health Literacy Deficits Among Patients With Chronic Kidney Disease

Inadequate health literacy in people with chronic kidney disease (CKD) is associated with poorer disease management and greater complications. There are limited data on the health literacy deficits of people with CKD. The aim of this study was to investigate the types and extent of health literacy deficits in patients with CKD using the multidimensional Health Literacy Management Scale (HeLMS) and to identify associations between patient characteristics and the domains of health literacy measured by the HeLMS. Invitations to participate were sent to patients with CKD attending the renal unit of a regional Australian hospital. These patients included predialysis, dialysis (peritoneal and hemodialysis), and kidney transplant patients. This study identified that inadequate health literacy—especially in the domains relating to attending to ones health needs, understanding health information, social support, and socioeconomic factors—was common. Male gender and education level were significantly associated with inadequate health literacy. The type and extent of health literacy deficits varied among CKD groups, and transplant patients had more deficits than other CKD patient groups. This study provides useful information for health professionals treating patients with CKD, especially with regard to the design of self-management interventions and health information.

Acid and stretch, but not capsaicin, are effective stimuli for ATP release in the porcine bladder mucosa: Are ASIC and TRPV1 receptors involved?

Stretch-evoked ATP release from the bladder mucosa is a key event in signaling bladder fullness. Our aim was to examine whether acid and capsaicin can also release ATP and to determine the receptors involved, using agonists and antagonists at TRPV1 and acid-sensing ion channels (ASICs). Strips of porcine bladder mucosa were exposed to acid, capsaicin or stretch. Strip tension was monitored. Bath fluid was collected for ATP measurement. Gene expression of ASICs and TRPV1 in porcine bladders was quantified using quantitative real-time PCR (qRT-PCR). Stretch stimulus (150% of original length) repeatedly and significantly increased ATP release to approximately 45 times basal release. Acid (pH 6.5, 6.0, 5.6) contracted mucosal strips and also increased ATP release up to 30-fold, without evidence of desensitization. Amiloride (0.3 μM) reduced the acid-evoked ATP release by approximately 70%, while capsazepine (10 μM) reduced acid-evoked ATP release at pH 6.0 and pH 5.6 (by 68% and 61%, respectively). Capsaicin (0.1-10 μM) was ineffective in causing ATP release, and also failed to contract porcine mucosal or detrusor strips. Gene expression for ASIC1, ASIC2, ASIC3 and TRPV1 was seen in the lateral wall, dome, trigone and neck of both detrusor and mucosa. In conclusion, stretch and acid induce ATP release in the porcine bladder mucosa, but capsaicin is ineffective. The pig bladder is a well-known model for the human bladder, however these data suggest that it should be used with caution, particularly for TRPV1 related studies.