Guiping Sui

Gap junctions and connexin expression in human suburothelial interstitial cells.

Objective  To determine whether suburothelial interstitial cells of the human bladder express gap junctions, and if so, to establish their extent and composition, using immunocytochemistry, confocal microscopy and electron microscopy.

Purinergic regulation of guinea pig suburothelial myofibroblasts

The Ca2+‐regulating and electrophysiological properties of guinea‐pig suburothelial myofibroblasts have been measured in order to investigate their potential role in the sensation of bladder fullness, due to their strategic position between the urothelium and afferent fibres. Previous work has shown that stretch of the bladder wall releases ATP. Cells that stain positively for vimentin were isolated. About 45% of cells (median membrane capacitance 13.3 pF) exhibited spontaneous depolarizations to about −25 mV with a physiological Cl− gradient (frequency 2.6 ± 1.5 min−1, duration 14.5 ± 2.2 s, n= 15). Under voltage‐clamp spontaneous inward currents (frequency 1.5 ± 0.2 min−1, duration 14.5 ± 7.0 s, n= 18) were recorded, with a similar reversal potential. The spontaneous currents were preceded by intracellular Ca2+ transients with a magnitude that was independent of membrane potential. All cells tested responded to ATP by generating an intracellular Ca2+ transient, followed by inward currents; the currents had a similar reversal potential and slope conductance to their spontaneous counterparts. ATP‐generated transients were mimicked by UTP and ADP but not by α,β‐methylene‐ATP (1–10 μm) or CTP (30 μm), indicating that ATP acts via a P2Y receptor. Transients were partially attenuated by 1 mm suramin but PPADS (80 μm) had no effect. These data indicate that ATP acts via a P2Y receptor, but responses were resistant to the P2Y1 antagonist MRS2179. ATP‐generated transients were abolished by intracellular perfusion with heparin and TMB‐8 indicating that IP3 was the intracellular second messenger. The reversal potentials of the spontaneous and ATP‐generated currents were shifted by about +45 mV by a 12‐fold reduction of the extracellular [Cl−] and the currents were greatly attenuated by 1 mm DIDS. No transients were generated on exposure to the muscarinic agonist carbachol. We propose that these cells may play a regulatory step in the sensation of bladder fullness by responding to ATP. The precise mechanism whereby they couple urothelial ATP release to afferent excitation is the next step to be elucidated.

Modulation of bladder myofibroblast activity: implications for bladder function.

Bladder suburothelial myofibroblasts may modulate both sensory responses from the bladder wall and spontaneous activity. This study aimed to characterize further these cells in their response to exogenous agents implicated in mediating the above activity. Detrusor strips, with or without mucosa, and isolated suburothelial myofibroblasts were prepared from guinea pig bladders. Isometric tension, intracellular Ca2+, and membrane current were recorded. Cell pairs were formed by pushing two cells together. Tension, intracellular Ca2+, and membrane potential were also recorded from bladder sheets using normal or spinal cord-transected (SCT) rats. Spontaneous contractions were greater in detrusor strips with an intact mucosa and were augmented by 10 μM UTP. ATP, UTP, or reduced extracellular pH elicited Ca2+ transients and inward currents (Erev −30 mV) in isolated cells. Capsaicin (5–30 μM) reduced membrane current (37 ± 12% of control) with minor effects on Ca2+ transients: sodium nitroprusside reduced membrane currents (40 ± 21% of control). Cell pair formation, without an increase in cell capacitance, augmented ATP and pH responses (180 ± 58% of control) and reduced the threshold to ATP and acidosis. Glivec (20–50 μM) reversibly blocked the augmentation and also reduced spontaneous activity in bladder sheets from SCT, but not normal, rats. Glivec also disrupted the spread of Ca2+ waves in SCT sheets, generating patterns similar to normal bladders. Suburothelial myofibroblasts respond to exogenous agents implicated in modulating bladder sensory responses; responses augmented by physical intercellular contact. The action of glivec and its selective suppression of spontaneous activity in SCT rats identifies a possible pathway to attenuate bladder overactivity.

Characterization of the purinergic receptor subtype on guinea‐pig suburothelial myofibroblasts

To identify particular purinoceptor subtypes by immunohistochemical labelling, as a layer of suburothelial myofibroblasts has been identified in the urinary bladder, and these cells respond to exogenous ATP by generating an intracellular Ca2+ transient, but the particular purinoceptor that responds to ATP is unclear.

The role of the L-type Ca2+ channel in refilling functional intracellular Ca2+ stores in guinea-pig detrusor smooth muscle

The transient rise of intracellular Ca2+ in detrusor smooth muscle cells is due to the release of Ca2+ from intracellular stores. However, it is not known how store refilling is maintained at a constant level to ensure constancy of the contractile response. The aim of these experiments was to characterise the role of L‐type Ca2+ channels in refilling. Experiments used isolated guinea‐pig detrusor myocytes and store Ca2+ content was estimated by measuring the magnitude of change to the intracellular [Ca2+] ([Ca2+]i) after application of caffeine or carbachol using epifluorescence microscopy. Membrane potential was controlled when necessary by voltage clamp. After Ca2+ stores were emptied they refilled with an exponential time course, with a time constant of 88 s. The value of the time constant was similar to that of the undershoot of [Ca2+]i following store Ca2+ release. The degree of store filling was enhanced by maintained depolarisation, or by transient depolarising pulses, and attenuated by L‐type Ca2+ channel antagonists. Inhibition of the sarcoplasmic reticular Ca2+‐ATPase prevented refilling. Reduction of the resting [Ca2+]i was accompanied by membrane depolarisation; under voltage clamp reduction of [Ca2+]i decreased the number and magnitude of spontaneous transient outward currents. Ca2+ release from intracellular stores, elicited by caffeine or carbachol, is independent of membrane potential under physiological conditions. However, store refilling occurs via Ca2+ influx through L‐type Ca2+ channels. Ca2+ influx is regulated by a feedback mechanism whereby a fall of [Ca2+]i reduces the activity of Ca2+‐activated K+ channels, causing cell depolarisation and an enhancement of L‐type Ca2+ channel conductance.

Aberrant Ca2+ oscillations in smooth muscle cells from overactive human bladders

Overactive bladder (OAB) syndrome is highly prevalent and costly, but its pathogenesis remains unclear; in particular, the origin of involuntary detrusor muscle activity. To identify the functional substrate for detrusor muscle overactivity, we examined intracellular Ca(2+) oscillations in smooth muscle cells from pathologically overactive human bladders. Basal cytoplasmic Ca(2+) concentration was elevated in smooth muscle cells from overactive bladders. Unprovoked, spontaneous rises of Ca(2+) were also identified. These spontaneous Ca(2+) oscillations were Ca(2+)-dependent, sensitive to L-type Ca(2+) channel antagonist verapamil and also attenuated by blocking SR Ca(2+) reuptake. The fraction of spontaneously active cells was higher in cells from overactive bladders and the magnitude of spontaneous Ca(2+) oscillations also greater. Spontaneous action potentials or depolarising oscillations were also observed, associated with Ca(2+) rise; with a higher percentage of cells from idiopathic OAB, but not in neurogenic OAB. Low concentrations of NiCl(2) attenuated both spontaneous electrical and Ca(2+) activation. This study provides the first evidence that spontaneous, autonomous cellular activity-Ca(2+) and membrane potential oscillations, originates from detrusor smooth muscle in human bladders, mediated by extracellular Ca(2+) influx and intracellular release. Such cellular activity underlies spontaneous muscle contraction and defective Ca(2+) activation contributes to up-regulated contractile activity in overactive bladders.

A description of Ca2+ channels in human detrusor smooth muscle

To characterize the Ca2+ channels in human detrusor smooth muscle and to investigate their contribution to spontaneous electrical activity.

Impedance measurements and connexin expression in human detrusor muscle from stable and unstable bladders

Three of this months Scientific Discovery papers highlight the importance of collaboration in delivering high quality scientific research. As scientific technology increases in power and cost, and specific areas of interest become more specialized, it is becoming more difficult to cover all aspects of a completeresearch story. Collaborating with other experts in the field or other fields, including industry, allows strong scientific proof to be generated for the hypothesis and aims. Building strong collaborative,inter‐disciplinary, multi‐institutional, international groups with academic and industrial partners is the way forward for all discovery. We look forward to publishing more of these collaborative papersin future issues of the BJU International.

The electrophysiological properties of cultured and freshly isolated detrusor smooth muscle cells.

PURPOSE We generated and characterized a convenient isolated cell model of human detrusor smooth muscle to understand mechanisms that may underlie detrusor instability and provide a suitable model to test potentially useful drugs. MATERIALS AND METHODS The electrophysiological properties of freshly isolated detrusor smooth muscle cells from human and guinea pig biopsies were compared with those undergoing cell culture to document in detail the changes that occur during primary culture and subsequent passages as well as the differences in the 2 species. RESULTS Resting electrical characteristics were changed in the cultured cells. Membrane potential was less negative (guinea pig -59 versus -42 mV.) and membrane resistance was less (138 versus 124.5 Omegacm.(2)). Regenerative action potentials were recorded in cultured and freshly isolated cells. In guinea pig cells the overall duration and initial rate of depolarization (upstroke) was slower in cultured than in freshly isolated cells, indicative of a decreased magnitude of ionic current in cultured cells. Human cells had a similar prolongation in culture but no decrease in the upstroke rate. Experiments with selective blockers indicated that depolarization is due to influx through L-type Ca2+ channels and repolarization occurred via Ca2+ dependent K+ channels in freshly isolated and cultured cells. No further changes to properties were observed in cells passaged up to 3 times from primary cultured cells. CONCLUSIONS Cell culture qualitatively preserves the electrophysiological properties of detrusor smooth muscle cells, although there is some decrease in channel density.

Characteristics of Spontaneous Activity in the Bladder Trigone

BACKGROUND During bladder filling, the trigone contracts help keep the ureteral orifices open and the bladder neck shut. The trigone generates spontaneous activity as well as responding to neuromuscular transmitters, but the relationship between these phenomena are unclear. OBJECTIVES To characterise the cellular mechanisms that regulate and modify spontaneous activity in trigone smooth muscle. DESIGN, SETTING, AND PARTICIPANTS Muscle strips from the superficial trigone of male guinea-pigs were used for tension experiments and immunofluorescent studies. MEASUREMENTS In isolated trigonal cells, intracellular Ca(2+) was measured by epifluorescence microscopy using the fluorescent Ca(2+) indicator Fura-2. RESULTS AND LIMITATIONS Spontaneous intracellular Ca(2+) transients and contractions were observed in trigonal single cells and strips and were significantly higher compared to the bladder dome. Ca-free superfusate and verapamil terminated spontaneity. T-type Ca(2+) channel block with NiCl(2) depressed slightly Ca(2+) transients but not spontaneous contractions. Neither the BK(Ca) channel blocker iberiotoxin nor the SK(Ca) channel blocker apamin had any effect on single cell activity. By contrast, the Cl(-) channel blocker niflumic acid attenuated significantly both Ca(2+) transients and muscle contractions. Agonist stimulation (carbachol, phenylephrine) up-regulated activity. Gap junction labelling (Cx43) was approximately 5 times denser in the trigone than in detrusor smooth muscle. The gap junction blocker 18-beta-glycyrrhetinic acid modulated spontaneous contractions in the trigone but not in the bladder dome. CONCLUSIONS Trigone myocytes employ membrane L-type-Ca(2+) channels and Cl(-) channels to generate spontaneous activity. Intercellular electrical coupling ensures its propagation and, thus, sustains contraction of the whole trigone.