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C H Fry

Publications and source records attributed to C H Fry.

At least 19 recordsLinked to original sources
PubMed2007

Ca2+ regulation in detrusor smooth muscle from ovine fetal bladder after in utero bladder outflow obstruction.

PURPOSE: We characterized intracellular Ca(2+) regulation in fetal bladders following outflow obstruction by examining the Ca(2+) response to agonists in smooth muscle cells. MATERIALS AND METHODS: Severe bladder outflow obstruction was induced in male fetal sheep by placing a urethral ring and urachal ligation midway through gestation at 75 days. Fetuses were examined 30 days after surgery. Intracellular Ca(2+) in single smooth muscle cells isolated from the bladder wall was measured with epifluorescence microscopy using fura-2(AM) during exposure to agonists, such as carbachol and adenosine triphosphate, and to other activators, such as caffeine and KCl. RESULTS: Detrusor smooth muscle cells from obstructed bladders had resting intracellular Ca(2+) similar to that in sham operated controls. The maximal response to carbachol was decreased following obstruction (p <0.05). Construction of dose-response curves also demonstrated higher EC(50) (p <0.05). However, these changes were not mirrored by caffeine evoked Ca(2+) release, which was not significantly different between the obstruction group and sham operated controls. Kinetic analysis of carbachol transients further revealed an attenuated maximal rate of increase in obstructed bladders (p <0.01). The magnitude of intracellular Ca(2+) to purinergic neurotransmitter adenosine triphosphate was also found to be smaller in cells from obstructed bladders (p <0.05), although transmembrane influx by high K depolarization was not significantly affected. CONCLUSIONS: Muscarinic and purinergic pathways were down-regulated in fetal detrusor muscle following outflow obstruction. These major functional receptors appeared to be more susceptible to obstruction than other Ca(2+) regulators. Their impairment may contribute to the compromised contractile function seen in in utero bladder outflow obstruction.

Animals↗
PubMed2006

T-type Ca2+ channels in non-vascular smooth muscles.

T-type Ca2+ current has been recorded in smooth muscle myocytes, and associated interstitial cells, isolated from the gastro-intestinal tract, urinary bladder, urethra, prostate gland, myometrium, vas deferens, lymphatic vessels and airways smooth muscle. By contrast, current through such channels has not been recorded from other tissues, such as the ureter. Whilst the properties of this Ca2+ current are similar in most of these cells, with respect to their voltage-dependence, ion selectivity and response to channel modulators, some differences have been recorded, most notably in the gastro-intestinal tract, and may demand a reappraisal of how a T-type Ca2+ current is characterised. The functions of such a current in different tissues remains uncertain. In most of smooth muscles discussed in this review, it is hypothesised that it underlies rhythmic or spontaneous electrical activity, especially in concert with other current-carrying systems, such as Ca2+-activated outward currents. Of equal interest is that the T-type Ca2+ channel may be a target for agents that modulate tissue function, especially in pathological conditions, or are the site of secondary effects of agents used in clinical medicine. For example, T-type Ca2+ channel modulators have been proposed to reduce overactive muscular activity in the gastro-intestinal or urinary tract, or function as tocolytic agents: and the action of volatile anaesthetics on them in airways smooth muscle requires consideration in their overall action.

Animals↗
PubMed2006

Ca2+ regulation in detrusor smooth muscle from developing fetal sheep bladders.

Sheep fetus is a useful model to study in utero bladder outflow obstruction but little is known about cell physiology of fetal bladders. To remedy this defect we have characterised intracellular Ca(2+) regulation in fetal sheep myocytes of different developmental ages. Fetal detrusor myocytes had a similar resting [Ca(2+)](i) to adult cells and exhibited transient [Ca(2+)](i) increases in response to carbachol, ATP, high-K, caffeine and low-Na. The carbachol transients were abolished by atropine and caffeine; the ATP response was blocked by alpha,beta-methylene ATP; high-K-evoked [Ca(2+)](i) rises were antagonised by verapamil. The maximal responses to carbachol, high-K, caffeine and low-Na in fetal cells were similar to those of adult counterparts, whilst the ATP response was smaller (p < 0.05). These variables were largely similar between the three gestational groups with the exception of ATP-induced response between early fetal and adult bladders (p < 0.05). Dose-response curves to carbachol demonstrated an increase of potency between mid-gestation and early adulthood (p < 0.05). These data show that muscarinic receptors coupled to intracellular Ca(2+) release, P2X receptor-linked Ca(2+) entry, depolarisation-induced Ca(2+) rise via L-type Ca(2+) channels, Na(+)/Ca(2+) exchange and functional intracellular Ca(2+) stores are all operational in fetal bladder myocytes. Whilst most of Ca(2+) regulators are substantially developed and occur at an early fetal age, a further functional maturation for cholinergic sensitivity and purinergic efficacy continues throughout to adulthood.

Adenosine Triphosphate↗
PubMed2005

Intracellular Ca2+ regulation in a human prostate stromal cell culture.

AIMS: Prostate stromal cell cultures are used in vitro to study the cellular pathophysiology of benign prostatic hyperplasia (BPH), but their functional properties are poorly understood. This study characterized intracellular Ca2+ ([Ca2+]i) regulation in a cultured cell line in comparison to freshly isolated cells, as a background to understanding contractile regulation and cellular proliferation in this tissue. METHODS: Prostate stromal cells were isolated from either PrS6 cell cultures, with an extended life span by transfection with the SV40 T-antigen, tsA58-U19, or freshly obtained transition zone prostate samples, primary cells. [Ca2+]i was measured in vitro with the indicator Fura-2 by epifluorescence microscopy. RESULTS: Phenylephrine, high-K+, and caffeine induced Ca2+-transients in primary cells (resting [Ca2+]i 94 +/- 8 nM, n = 29; peak 193 +/- 26 nM, n = 19). In PrS6 cells resting [Ca2+]i was 96 +/- 8 nM (n = 78) and in 34 of these 78 cells, 30 microM phenylephrine increased [Ca2+]i to 296 +/- 28 nM. 5-methyl-urapidil (10-30 microM) inhibited this response in 10 of 16 cells. Spontaneous Ca2+-transients were also observed in 91% of phenylephrine-responsive cells, but in only 20% of non-responsive cells (P < 0.01). Ca2+-transients were also induced by high-K+ solution, and 20 mM caffeine. The latter abolished the response to subsequent phenylephrine application. Depletion of intracellular Ca2+ stores by caffeine or restoration from a Ca2+-free superfusate caused a substantial rise of [Ca2+]i. CONCLUSIONS: PrS6 prostate stromal cells express functional alpha1-adrenoceptors associated with spontaneous intracellular Ca2+-transients. They exhibit functional Ca2+ channels, intracellular Ca2+ stores, and Ca2+ entry induced by store depletion. Stromal cultures can therefore be used to characterize the cellular physiology of prostate stromal cell contraction and proliferation.

Adrenergic alpha-Agonists↗
PubMed2004

Purinergic regulation of guinea pig suburothelial myofibroblasts.

The Ca(2+)-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 Ca(2+) transients with a magnitude that was independent of membrane potential. All cells tested responded to ATP by generating an intracellular Ca(2+) 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 alpha,beta-methylene-ATP (1-10 microm) or CTP (30 microm), indicating that ATP acts via a P2Y receptor. Transients were partially attenuated by 1 mm suramin but PPADS (80 microm) had no effect. These data indicate that ATP acts via a P2Y receptor, but responses were resistant to the P2Y(1) antagonist MRS2179. ATP-generated transients were abolished by intracellular perfusion with heparin and TMB-8 indicating that IP(3) 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.

Animals↗
PubMed2004

Ca2+ currents in smooth muscle cells isolated from human prostate.

BACKGROUND: The objective of the investigation was to demonstrate the presence of voltage-dependent Ca2+ current in human prostate smooth muscle cells and to determine their biophysical characteristics. METHODS: Prostate smooth muscle cells were isolated from biopsy samples of human prostates obtained from prostatectomy specimens or TURP chips. Electrophysiological recordings were made under current- or voltage-clamp using patch-type electrodes. RESULTS: The average resting potential of prostate myocytes was 63 +/- 11 mV and action potentials (APs) could be elicited when K+ currents were blocked. With K-filled electrodes inward current was followed by a large outward component. When K+ currents were blocked a large Ca2+-sensitive inward current was measured. The inward current could be divided into two components, a fraction blocked by 30 microM verapamil and another by 20 microM NiCl2. CONCLUSIONS: Based on the sensitivity to antagonists and holding potential both L-type and T-type Ca2+ channels were identified in human prostate smooth muscle.

Biopsy↗
PubMed2004

Recent advances in detrusor muscle function.

Contractile activation of detrusor smooth muscle is initiated by the release of transmitters from motor nerves. Acetylcholine is a ubiquitous transmitter, as also is adenosine triphosphate (ATP) in many animal bladders and in people from several patient groups with pathological bladder function. In recent years there has been progress in explaining several cellular mechanisms that link transmitter release to contraction and these will be considered. The lifetime of ATP in the neuromuscular junction is finite and broken down ultimately to adenosine, which can exert modulatory control of contractile activation. Adenosine depresses nerve-mediated contractions and two sites of action have been proposed: an action on the motor nerves via A receptors to depress further transmitter release and a less well-defined depressant effect on the detrusor muscle. The Ca2+ ions that activate the contractile proteins are derived from intracellular stores, which releases their content via IP receptor activation and Ca2+-induced Ca2+ release. Filling of the stores in the rest interval is mediated via transmembrane flux of Ca2+through Ca2+ channels. Activation of the channels is regulated by the level of the intracellular [Ca2+], via activation and inactivation of Ca2+-sensitive K channels. Thus, Ca2+ store filling is regulated by intracellular [Ca2+] via a negative feedback process. The presence and physiological function of spontaneous contractions in detrusor remain contentious and little is known about their origin. One possibility is that they originate from random Ca2+ sparks, i.e. localized transient increases of [Ca2+] that may eventually progress to generate a cellular Ca2+ transient. Observations by confocal microscopy have revealed the presence of such sparks, especially near the cell membrane, and thus provide a cellular basis for spontaneous contractions. Finally, the questions arises as to whether detrusor smooth muscle is a functional syncitium. The demonstration of small gap junctions by electron microscopy and the demonstration of the gap junction protein connexin45 indicate that the muscle mass may indeed be functionally connected. The implications regarding the spread of excitation are discussed.

Animals↗
PubMed2004

Electrical characteristics of suburothelial cells isolated from the human bladder.

PURPOSE: We measured the membrane electrical characteristics as well as the response to adenosine triphosphate of cells isolated from the suburothelial layer of the bladder. MATERIALS AND METHODS: Suburothelial cells were isolated from biopsy samples of human bladder by collagenase disruption. Electrophysiological measurements were done under current and voltage clamp to record membrane potential and ionic currents using patch pipettes with a K+ based filling solution. Intracellular [Ca2+] was measured with Fura-2. RESULTS: Cells were different from epithelial cells by their spindle-shaped appearance with projections at either end. The cells stained for vimentin but epithelial and smooth muscle cells did not. The cells had small membrane capacitance (27 +/- 16 pF) and a specific membrane resistance of 90 +/- 48 x 10(9) Omega cm2. Average membrane potential was -63 +/- 14 mV but cells showed spontaneous spikes or random fluctuations of membrane potential. A small net inward current was superimposed by a larger outward current. Inward current was attenuated by the removal of extracellular Ca. Outward current showed large spontaneous fluctuations and was greatly decreased by 30 mM tetraethyl ammonium chloride. Adenosine triphosphate (30 to 100 microM) elicited an inward current of about 50 pA and large intracellular Ca2+ transients. CONCLUSIONS: These cells are electrically active which, in conjunction with the previous observation of connexin 43 labeling, suggests that they could act as an electrical network. A quantitative model of voltage distribution in such a network after the generation of inward current suggests that individual cells could not act as pacemakers, but rather a group of simultaneously activated cells could exert a peripheral excitatory effect that would amplify the magnitude of the original response. The implications of this in terms of bladder sensation are discussed.

Adenosine Triphosphate↗
PubMed2004

Characterization of the control of intracellular [Ca2+] and the contractile phenotype of cultured human detrusor smooth muscle cells.

PURPOSE: We measured the functional properties of cultured human detrusor myocytes with respect to their ability to regulate their intracellular [Ca2+] and generate force in collagen matrices. MATERIALS AND METHODS: Human detrusor biopsies were dissociated into single cells by collagenase treatment and used immediately or cultured in D-valine medium and subsequently used after culture trypsinization. Intracellular [Ca2+] was measured in Fura-2 loaded myocytes. Cell force development was measured by incorporating cells into a collagen gel and attaching it to an isometric strain gauge. RESULTS: Carbachol was equally effective in generating Ca transients in freshly isolated and cultured cells. Carbachol potency (pEC50) and the magnitude of Ca2+ transients were similar. Adenosine triphosphate potency was decreased in cultured cells and Ca2+ transients showed properties consistent with a purinoceptor shift from a purinergic subtype. Temporal restitution of Ca2+ transients was similar in the 2 groups, indicative of retained intracellular Ca2+ stores in cultured cells. Cultured cells (approximately 10(6)) embedded in collagen gel generated a force about 10 times greater than that generated by gel alone. The cell dependent force could be further increased by adding carbachol. CONCLUSIONS: Cultured cells retain the ability to generate agonist induced intracellular Ca2+ transients. There was no evidence that the cell culture altered the properties of muscarinic receptors, although purinoceptor mediated properties were altered. Restitution experiments indicated that functional intracellular Ca2+ stores were retained in cultured cells. Cultured cells also retained a contractile phenotype, especially in response to carbachol. The magnitude of force was attenuated, which may be a function of the biomechanical properties of the gel used to embed the cells.

Calcium↗
PubMed2003

Neurotransmission and viscoelasticity in the ovine fetal bladder after in utero bladder outflow obstruction.

Fetal bladder outflow obstruction, predominantly caused by posterior urethral valves, results in significant urinary tract pathology; these lesions are the commonest cause of end-stage renal failure in children, and up to 50% continue to suffer from persistent postnatal bladder dysfunction. To investigate the physiological development of the fetal bladder and the response to urinary flow impairment, we performed partial urethral obstruction and complete urachal ligation in the midgestation fetal sheep for 30 days. By electrical and pharmacological stimulation of bladder strips, we found that muscarinic, purinergic, and nitrergic mechanisms exist in the developing fetal bladder at this gestation. After bladder outflow obstruction, the fetal bladder became hypocontractile, producing less force after nerve-mediated and muscarinic stimulation with suggested denervation, and also exhibited greater atropine resistance. Furthermore, fetal bladder urothelium exerted a negative inotropic effect, partly nitric oxide mediated, that was not present after obstruction. Increased compliance, reduced elasticity, and viscoelasticity were observed in the obstructed fetal bladder, but the proportion of work performed by the elastic component (a physical parameter of extracellular matrix) remained the same. In addition to denervation, hypocontractility may result from a reduction in the elastic modulus that may prevent any extramuscular components from sustaining force produced by detrusor smooth muscle.

Adenosine↗
PubMed2003

Role of the T-type Ca2+ current on the contractile performance of guinea pig detrusor smooth muscle.

AIMS: The importance of the T-type Ca(2+) current in determining detrusor contractile function was investigated by using guinea pig muscle in vitro. METHODS: NiCl(2) (200 microM) was used to block selectively the T-type Ca(2+) current, and 20 microM verapamil was used to block the L-type Ca(2+) current in this tissue. The selectivity of these agents at such concentrations has been previously demonstrated. RESULTS: In normal extracellular solution (4 mM KCl) 200 microM NiCl(2) and 20 microM verapamil reduced electrically stimulated contractions by 17 +/- 6% and 65 +/- 10%, respectively. At high concentrations of the two agents, the contraction was completely abolished by NiCl(2) but by only 74 +/- 18% in the case of verapamil; this finding suggests that NiCl(2) has additional negative inotropic actions at higher concentrations. Carbachol and KCl contractures were attenuated to a similar extent to that of electrically stimulated contractions by NiCl(2) and verapamil, which suggests that they act on the muscle rather than the motor nerve. The dependence of the membrane potential on the relative ability of NiCl(2) and verapamil to attenuate the contraction was tested by varying the extracellular [KCl], [KCl](o). Varying [KCl](o) between 2 and 10 mM depolarised detrusor myocytes from (-65.1 +/- 4.7 mV to -42.7 +/- 4.0 mV (a slope of 32 mV per 10-fold change of [KCl](o)). In low [KCl](o),blockade by NiCl(2) was more effective and that of verapamil less effective; at high [KCl](o), the reverse potency was recorded. CONCLUSIONS: The data are consistent with the hypothesis that Ca(2+) influx through both T-type and L-type Ca(2+) channels determines the contractile status of detrusor smooth muscle and that T-type channel activity is more important at membrane potentials near the resting level. A significant role for T-type channel activity in the resting state was evident in that spontaneous contractions were attenuated to a greater extent than evoked contractions.

Animals↗
PubMed2003

Electromyographic detection of purinergic activity in Guinea pig detrusor smooth muscle.

PURPOSE: We recorded nerve mediated extracellular electrical activity from guinea pig detrusor smooth muscle strips using suction electrodes and determined the electrophysiological origins of this signal and its relationship to contractile activity. MATERIALS AND METHODS: Mucosa-free detrusor strips were prepared from male guinea pigs sacrificed under Home Office license, physiologically superfused, attached to a pressure transducer and electrically stimulated (0.1 millisecond pulses). Electrical signals recorded using a bipolar reversible suction electrode were processed and recorded simultaneously with changes in strip tension. The effect of superfusion with alpha, beta-methylene adenosine triphosphate (ATP), atropine, extracellular [CaCl(2)] depletion and pharmacological Ca2+ channel blockade on the electrical and mechanical signals was determined. RESULTS: A biphasic electrical signal was consistently recorded from 37 detrusor strips. The signal was sensitive to graded reduction in [CaCl(2)] of the superfusate and abolished by tetrodotoxin in 7 preparations. The signal was also abolished in 12 preparations by alpha, beta-methylene ATP in association with an attenuated contraction but not significantly reduced in amplitude (p = 0.77) despite a significant reduction in tension with atropine (mean plus or minus SD 74% +/- 14% of control, p <0.001). The signal was attenuated to a mean maximum of 9% +/- 3% of control by pharmacological Ca2+ channel blockade and the remaining signal was abolished by alpha, beta-methylene ATP. CONCLUSIONS: The extracellular electrical signal recorded from guinea pig detrusor strips using suction electrodes originates from a purinergic mechanism. Although an atropine sensitive component may be present, the signal does not depend on cholinergic neuromuscular transmission and would not be expected to be generated by normal human detrusor. Provided that the electrophysiological basis of purinergic neurotransmission in guinea pig and human bladders is similar suction electrodes may be a valuable tool with which to evaluate in vitro and clinically by electromyography the pathological purinergic neuromuscular transmission that can be expressed in addition to normal cholinergic mechanisms in detrusor from dysfunctional human bladders.

Adenosine Triphosphate↗
PubMed2003

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

UNLABELLED: Three of this month's 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. OBJECTIVES: To test the hypothesis that intercellular electrical coupling is altered in human detrusor smooth muscle from patients with unstable bladders. MATERIALS AND METHODS: Human detrusor biopsy samples were obtained from patients with stable and unstable bladders. Intracellular electrical impedance was measured with alternating current (20 Hz-300 kHz) across the ends of detrusor strips in an oil-gap, after correcting for extracellular space resistance. Gap junctions were identified by localization of connexins (Cx), specifically Cx45, Cx43 and Cx40 transcripts, using immunoconfocal microscopy. RESULTS: Total intracellular resistivity was greater in strips from unstable than from stable bladders (median 1246 vs 817 Omega.cm). The increase was attributed to an increase in junctional resistance; cytoplasmic resistance was unchanged. Cx43 was localized to a submucosal layer and to connective tissue; Cx40 label was confined to endothelial cells of blood vessels. Cx45 labelling was localized to detrusor bundles and appeared to be less marked in samples from unstable bladders. Semi-quantitative analysis of Northern blots showed that Cx45 expression in unstable was less than that in stable bladders. CONCLUSIONS: These data suggest that intercellular coupling is reduced in detrusor from unstable bladders. Cx45 was localized to the detrusor layer, with Cx 43 more evident in the suburothelial mucosa. Cx45 labelling was less intense in detrusor samples from unstable bladders. These results are consistent with reduced gap junction coupling in detrusor from unstable bladders.

Blotting, Northern↗
PubMed2003

A description of Ca2+ channels in human detrusor smooth muscle.

OBJECTIVE: To characterize the Ca2+ channels in human detrusor smooth muscle and to investigate their contribution to spontaneous electrical activity. MATERIALS AND METHODS: Isolated human detrusor smooth muscle myocytes were used to measure ionic currents under voltage-clamp or membrane potential under current-clamp. Membrane potential oscillations were analysed in terms of oscillation frequency and amplitude using fast Fourier transforms. RESULTS: Under voltage-clamp an inward current dependent on extracellular Ca2+ was recorded using Cs+-filled patch electrodes. The current could be separated into two components on the basis of their sensitivity to Ni2+, verapamil or nicardipine, and their dependence on holding and clamp potential. A Ni2+-sensitive component activated over a relatively negative range of potentials (-60 to -20 mV) comprised about a third of the total current and was designated a T-type Ca2+ current. A verapamil/nicardipine-sensitive component, activated at more positive potentials, was designated an l-type Ca2+ current. Using K+-based filling solutions spontaneous transient outward currents were recorded that had the characteristics of current flow through BK channels. Membrane potential oscillations, under current-clamp increased in frequency but not amplitude as the mean membrane potential was made less negative. The voltage-dependence of oscillation frequency was similar to that of the l-type, but not T-type, Ca2+ current activation curve. Furthermore oscillation frequency was slowed by verapamil but not Ni2+. CONCLUSION: The study showed, for the first time, the presence of both T- and L-type Ca2+ channels in human detrusor smooth muscle; we propose a role for these channels in spontaneous activity. The results suggest that the L-type Ca2+ current can control membrane potential oscillation frequency. The significance of this finding for spontaneous contractions is discussed.

Calcium Channels, L-Type↗
PubMed2002

The role of the L-type Ca(2+) channel in refilling functional intracellular Ca(2+) stores in guinea-pig detrusor smooth muscle.

The transient rise of intracellular Ca(2+) in detrusor smooth muscle cells is due to the release of Ca(2+) 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 Ca(2+) channels in refilling. Experiments used isolated guinea-pig detrusor myocytes and store Ca(2+) content was estimated by measuring the magnitude of change to the intracellular [Ca(2+)] ([Ca(2+)](i)) after application of caffeine or carbachol using epifluorescence microscopy. Membrane potential was controlled when necessary by voltage clamp. After Ca(2+) 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 [Ca(2+)](i) following store Ca(2+) release. The degree of store filling was enhanced by maintained depolarisation, or by transient depolarising pulses, and attenuated by L-type Ca(2+) channel antagonists. Inhibition of the sarcoplasmic reticular Ca(2+)-ATPase prevented refilling. Reduction of the resting [Ca(2+)](i) was accompanied by membrane depolarisation; under voltage clamp reduction of [Ca(2+)](i) decreased the number and magnitude of spontaneous transient outward currents. Ca(2+) release from intracellular stores, elicited by caffeine or carbachol, is independent of membrane potential under physiological conditions. However, store refilling occurs via Ca(2+) influx through L-type Ca(2+) channels. Ca(2+) influx is regulated by a feedback mechanism whereby a fall of [Ca(2+)](i) reduces the activity of Ca(2+)-activated K(+) channels, causing cell depolarisation and an enhancement of L-type Ca(2+) channel conductance.

Animals↗
PubMed2002

The contractile potency of adenosine triphosphate and ecto-adenosine triphosphatase activity in guinea pig detrusor and detrusor from patients with a stable, unstable or obstructed bladder.

PURPOSE: We compared the potency of adenosine triphosphate (ATP) and its nonhydrolyzable analogue alpha,beta-methylene ATP for generating contractions in human detrusor smooth muscle from patients with a stable, unstable and obstructed bladders. The different ATP potencies were compared with the ecto-adenosine triphosphatase (ATPase) of these samples. MATERIALS AND METHODS: Contractile experiments were done in vitro by superfusing samples with purines and dose-response curves were generated. Ecto-ATPase activity was measured from the rate of ATP hydrolysis sensitive to the ecto-ATPase inhibitor ARL 67156 with a luciferin-luciferase assay. RESULTS: ATP generated contractions with a mean EC50 of 933 microM. in tissue from stable bladders and was significantly more potent in tissue from unstable and obstructed bladders (EC50 141 and 172 microM., respectively). alpha,beta-methylene ATP was more potent in tissue from stable and unstable bladders (mean combined EC50 3 microM.). In guinea pig detrusor the mean EC50 for ATP and alpha,beta-methylene ATP was 138 and 5.5 microM., respectively. Mean total ATPase activity in unstable bladder biopsies plus or minus standard deviation was about 50% of that in stable bladder biopsies (2.54 +/- 1.50 versus 1.37 +/- 0.46 nmol. per second per mg. protein ). The ARL 67156 sensitive fraction was also significantly less in samples from unstable compared with stable bladders (mean 0.94 +/- 0.41 versus 0.36 +/- 0.26 nmol. per second mg. protein ). CONCLUSIONS: The greater potency of ATP for generating contractions in detrusor from unstable bladders may be due to reduced extracellular hydrolysis, allowing purine greater access to detrusor smooth muscle. This finding may explain atropine resistant purine based contractions in detrusor from unstable bladders.

Adenosine Triphosphatases↗
PubMed2002

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. MATERIALS AND METHODS: Bladder tissue was obtained at cystectomy; the tissue was: (i) frozen for cryosectioning and Northern blot analysis; (ii) fixed and embedded for standard thin-section electron microscopy; and (iii) processed using low-denaturation conditions in Lowicryl for immunogold-label electron microscopy. Cryosections were immunofluorescently labelled using antibodies against connexins 43, 40 and 45, vimentin, desmin and c-Kit ligand, and examined by confocal microscopy. Double labelling was used to determine the spatial relationship of labelling for connexin43 with that of vimentin and desmin. Thin-section electron microscopy was used to investigate interstitial cell ultrastructure and permit unequivocal identification of gap junctions, and immunogold labelling of Lowicryl sections was applied to localize connexin43. RESULTS: Immunoconfocal microscopy showed prominent labelling for the gap junction protein, connexin43, in a suburothelial band of cells that was also strongly positive for vimentin. The connexin43/vimentin-positive cells showed only weak labelling for desmin and c-Kit ligand, and were immunonegative for connexins 40 and 45. Northern blotting showed a corresponding abundance of connexin43 transcript in the mucosal layer but not the detrusor layer of the bladder wall. Electron microscopy revealed abundant gap junctions, recognized by their pentalaminar structure, between the cell processes of interstitial cells in the suburothelial zone. That these interstitial cell gap junctions were the source of the connexin43 immunolabelling observed by immunoconfocal microscopy was confirmed by immunogold labelling in sections of Lowicryl-embedded tissue examined by electron microscopy. CONCLUSION: A network of interstitial cells, extensively linked by connexin43-containing gap junctions, is located beneath the urothelium in human bladder. As gap junctions provide pathways for direct cell-to-cell communication, the interstitial cellular network may operate as a functional syncytium, integrating signals and responses in the bladder wall.

Blotting, Northern↗