Changhao Wu

A QUANTITATIVE STUDY OF ATROPINE-RESISTANT CONTRACTILE RESPONSES IN HUMAN DETRUSOR SMOOTH MUSCLE, FROM STABLE, UNSTABLE AND OBSTRUCTED BLADDERS

PURPOSE The objective of the study was to quantify in vitro the magnitude of atropine-resistant contractions using human detrusor samples and to determine the cellular processes underlying these contractions. MATERIALS AND METHODS Isometric contractile responses were measured in isolated strips of human detrusor muscle obtained from patients with i) stable, ii) unstable or iii) obstructed bladders. Preparations were electrically stimulated or exposed to carbachol and ATP in the superfusate. RESULTS Force-frequency curves were shifted to the right in samples from unstable and obstructed bladders. These same tissue groups also showed significant atropine-resistant contractions which were abolished by the neurotoxin TTX, or the non-hydrolysable ATP analog, alpha,beta-methylene ATP, suggesting that these contractions were mediated by neurally released ATP. Sub-division of the patient group with unstable bladders demonstrated that those with neuropathic instability did not show atropine-resistance, whereas those with idiopathic instability or secondary instability after obstruction did show atropine-resistant contractions. The potency of carbachol in generating a contracture was significantly greater than ATP (mean EC50 0.65 microM and 151 microM respectively) however, for each agonist there was no difference in potency between the three patient groups. Direct muscle excitability was similar in all three patient groups. CONCLUSIONS It is concluded that purinergic, atropine-resistant contractions are present in some types of dysfunctional bladder, and these are not caused by a differential sensitivity of the muscle to ATP and cholinergic agonists.

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.

A COMPARISON OF THE MODE OF ACTION OF ATP AND CARBACHOL ON ISOLATED HUMAN DETRUSOR SMOOTH MUSCLE

PURPOSE The objectives of the study were: i) to examine the ability of carbachol and ATP to raise intracellular [Ca2+] in isolated detrusor myocytes; ii) to determine the origin of the intracellular Ca2+ and iii) to address the question of whether the appearance of purinergic contractions in detrusor from unstable and obstructed human bladders is reflected in the sensitivity of the cell to the two agonists. MATERIALS AND METHODS Intracellular Ca2+ transients generated by extracellular ATP and carbachol were recorded from isolated human detrusor myocytes. Cells were dissociated by collagenase disruption of the biopsy. Intracellular Ca2+ was measured by epifluorescence microscopy using Fura-2 and electrophysiological recordings were made with patch electrodes. RESULTS In cells from stable bladder biopsies the half-maximal concentrations (EC50) for ATP and carbachol to generate Ca2+ transients were 0.10 and 0.25 microM respectively. With cells from unstable bladders the EC50 values for both agonists and the magnitude of the Ca2+ transients were not significantly different from those obtained in cells from normal bladders. The transient in ATP was preceded by a transient depolarisation generated by a large inward current. The carbachol-Ca2+ transient was independent of changes to membrane potential, except in a subset of cells where complex membrane potential changes followed the rise of intracellular Ca2+. The ATP-Ca2+ transient was partially abolished by nicardipine and completely abolished by zero-Ca solutions, the carbachol-Ca2+ transient was unaffected by nicardipine and less completely attenuated by zero-Ca solutions. Prior exposure to caffeine suggested that the carbachol-Ca2+ transient, but not the ATP-Ca2+ transient, originated from intracellular stores. CONCLUSIONS It is concluded that both agonists are equipotent in increasing intracellular Ca2+, but by different routes. The generation of purinergic contractions in detrusor from unstable bladder is not due to altered sensitivities of the detrusor myocyte to ATP or cholinergic agonists.

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.

Inward calcium currents in cultured and freshly isolated detrusor muscle cells : Evidence of a T-type calcium current

PURPOSE We carefully examined the possible routes of Ca2+ influx, and determined whether cultured cells retain Ca2+ channels and whether the culturing process changes their properties. MATERIALS AND METHODS Inward currents were measured under voltage clamp in freshly isolated cells and myocytes from confluent cell cultures of detrusor smooth muscle. RESULTS In guinea pig and human cells mean peak inward current density plus or minus standard deviation decreased significantly in cell culture (2.0 +/- 0.9 versus 4.5 +/- 2.2 pA.pF.(-1)) but there was no species variation. In primary cultured and passaged guinea pig cells an inward current was identified as L-type Ca2+ current. In freshly isolated cells another component to the inward current was identified that was insensitive to 20 micromol. l(-1) verapamil and 20 to 50 micromol. l(-1) cadmium chloride but abolished by 100 micromol. l(-1) nickel chloride and identified as T-type Ca2+ current. In addition, total inward current was greater at a holding potential of -100 than -40 mV., also indicating a component of current activated at negative voltage. Steady state activation and inactivation curves of the net inward current were also compatible with a single component in cultured cells but a dual component in freshly isolated cells. The action potential was completely abolished in cultured cells by L-type Ca2+ channel blockers but incompletely so in freshly isolated cells. Outward current depended strongly on previous inward current, suggesting a predominant Ca2+ dependent outward current. CONCLUSIONS In freshly isolated guinea pig cells T and L-type Ca2+ current is present but T-type current is absent in confluent cultures.

The cellular basis of contraction in human detrusor smooth muscle from patients with stable and unstable bladders

Studying the cellular physiology of human detrusor muscle obtained from patients with normally functioning bladders and comparing it with that of detrusor muscle from patients with unstable bladders may help identify potential targets for drug therapy in patients with abnormal bladder function. Considerable advances have been made in the understanding of the cellular processes that result in contraction and relaxation of detrusor smooth muscle, particularly in the role and modulation of calcium. Several changes in these cellular mechanisms that impair normal function have been observed in detrusor muscle from patients with unstable bladders. Whether these changes represent primary causes of bladder dysfunction or whether they are secondary to bladder dysfunction remains to be determined. Nevertheless, the identification of specific cellular lesions in bladder dysfunction presents a novel approach to identification of drug targets and potential treatment modalities.

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.