James I. Gillespie

Autonomous Activity in the Isolated Guinea Pig Bladder

Phasic changes in pressure have been reported to occur in the bladder which are not associated with micturition. Spontaneous intravesical pressure changes can be recorded from bladders in vitro or bladders in vivo isolated from the central nervous system suggesting that the bladder itself is capable of autonomous activity. Experiments using isolated cells and muscle strips indicate that the smooth muscle can generate spontaneous activity. Whether this is the origin of phasic changes in the intact organ remains unknown. The present study set out to establish the presence and characteristics of autonomous activity in the isolated guinea pig bladder. Multiple‐point motion analysis and concurrent intravesical pressure recording were used to identify and quantify spontaneous and evoked activity. Highly complex autonomous activity was observed in unstimulated bladders. This activity comprised localised micro‐contractions in single or multiple discrete regions, waves of activity and micro‐stretches. Low‐amplitude phasic ‘micro‐transients’ were seen in the intravesical pressure trace in association with micro‐contractions. Incremental increases in the intravesical volume recruited additional areas of activity. Atropine and tetrodotoxin had no effect on the micro‐transients or micro‐contractions. Exposure to the muscarinic agonist arecaidine (10‐300 nM) initially increased the incidence of micro‐contractions which subsequently became co‐ordinated into phasic pressure rises and contraction waves, interspersed with periods of total quiescence. The findings describe the generation and co‐ordination of autonomous activity in the bladder wall and also demonstrate complex phasic activity. This approach has shown the importance of assessing the integrative properties of the entire organ in studies of the physiology and patho‐physiology of the bladder.

Agonist- and nerve-induced phasic activity in the isolated whole bladder of the guinea pig: evidence for two types of bladder activity.

Spontaneous localised propagating waves of contraction and localised stretches have been reported to occur in the isolated whole bladder of the guinea pig. The physiological role and the cellular processes underlying these events are unknown. In order to gain insight into the mechanisms generating this complex activity, experiments were performed to examine and compare the responses of the whole bladder preparation to (i) the muscarinic agonists carbachol and arecaidine, (ii) the nicotinic ligand lobeline and (iii) nerve stimulation. High concentrations of the muscarinic agonists (>3 μM) induced a slow rise in intra‐vesical pressure upon which were superimposed pressure transients, while low concentrations (< 300 nM) induced only phasic rises in pressure. One interpretation of these data is that there are two separate mechanisms activated by muscarinic agonists: one generating contracture and the other phasic activity. Immunocytochemical staining revealed M3 muscarinic receptors on smooth muscle cells within trabeculae and a second population of positive cells in the sub‐urothelial layer. This observation raises the possibility that the actions of muscarinic agonists are a consequence of activating different cell types. Lobeline (1‐60 μM) activated phasic contractions but did not cause a rise in basal pressure. Atropine did not inhibit the lobeline‐induced responses but abolished the muscarinic responses. Also, hexamethonium or tetrodotoxin did not affect the lobeline‐induced responses. These observations suggest that the mechanism generating phasic activity is activated by a nicotinic stimulus that does not involve ganglia, nerves or the neuromuscular junction. Stimulation of the bladder nerve at frequencies between 20 and 30 Hz for 5 s resulted in a rapid rise in intra‐vesical pressure. Prolonged nerve stimulation (10‐200 s) at frequencies between 1 and 10 Hz activated phasic rises in pressure. Low frequency nerve stimulation increased the frequency of agonist‐induced phasic activity. Thus, nerve stimulation can also produce two forms of activity and low frequency stimulation can augment the processes generating phasic activity. These observations suggest that there are two distinct types of bladder activity: global contractions involving most of the bladder wall and phasic contractions comprising propagating waves of contraction. The mechanisms generating these contractile events appear to be different and they may involve cells located in different regions of the bladder. The nature of these mechanisms and their possible physiological significance is discussed.

Scanning electrochemical microscopy at the surface of bone-resorbing osteoclasts : Evidence for steady-state disposal and intracellular functional compartmentalization of calcium

Osteoclast resorptive activity occurs despite the presence of extremely high levels of ionized calcium ([Ca2+]) within the osteoclast hemivacuole, which is generated as a by‐product of its resorptive activity. Previous in vitro observations have shown that increases in extracellular [Ca2+] ([Ca2+]e) in the surrounding medium can inhibit the osteoclast resorptive activity. Therefore, it has been suggested that the osteoclast acts as a “sensor” for [Ca2+]e, and that high [Ca2+]e leads to an increase in intracellular [Ca2+] ([Ca2+]i), thereby inhibiting osteoclasts in a negative feedback manner. In this report we have carried out an experimental and theoretical analysis of calcium disposal during osteoclast activity to evaluate how in vitro models relate to in vivo osteoclast activity, where it is possible that high [Ca2+]e may be present in the hemivacuole but not over the nonresorbing surface of the cell. Scanning electrochemical microscopy (SECM) studies of [Ca2+] and superoxide anion () generation by bone‐resorbing osteoclasts on the surface of a bovine cortical bone slice were compared with microspectofluorometric measurements of the levels of [Ca2+]i in single osteoclasts and the effect of [Ca2+]i on various aspects of osteoclast function. The generation of by the osteoclasts has been shown to be positively correlated with osteoclast resorptive function and can therefore serve as an index of acute changes in osteoclast activity. The SECM of bone‐resorbing osteoclasts at the surface of a bone slice revealed a continuous steady‐state release of Ca2+. Even after prolonged incubation lasting 3 h the near‐surface [Ca2+]e in the solution above the cell remained <2 mM. The SECM real‐time measurement data were consistent with the osteoclast acting as a conduit for continuous Ca2+ disposal from the osteoclast‐bone interface. We conclude that the osteoclast distinguishes [Ca2+]e in the hemivacuole and in the extracellular fluid above the cell which we denote [Ca2+]e′. We found that an increase in [Ca2+]i may be associated with activation; inhibition; or be without effect on generation, bone‐matrix, or bone resorption. Similarly, osteoclast adhesion and bone‐resorbing activity was affected by [Ca2+]e′ but showed no correlation with [Ca2+]i. The data suggest the existence of functional compartmentalization of [Ca2+]i within the osteoclast, where elevated calcium may have an inhibitory, excitatory, or no effect on the overall osteoclast activity while exerting a selective effect on different functional modalities. These observations lead to the conclusion that far from being inhibited by Ca2+ generated, the osteoclast by virtue of the observed functional compartmentalization is highly adapted at carrying out its activity even when the level of [Ca2+] in resorptive lacunae is elevated.

Phasic non-micturition contractions in the bladder of the anaesthetized and awake rat

To characterize the contractile activity that occurs in the bladder during the filling phase of the micturition cycle (non‐micturition contractions, NMCs), which generate transient rises in intravesical pressure not associated with urine flow.

The production of nerve growth factor by human bladder smooth muscle cells in vivo and in vitro.

Objectiveu2002To measure the concentrations of nerve growth factor (NGF) in tissue biopsies taken from subjects with a normal bladder and from patients diagnosed to have idiopathic detrusor instability (associated with a reduction in the density of motor nerves), and to use an in vitro model to study the mechanisms of NGF expression.

Interstitial cells and phasic activity in the isolated mouse bladder

To describe the distribution of interstitial cells (ICs, defined as cells which show an increase in cGMP in response to nitric oxide, NO) in the isolated mouse bladder, and changes in phasic contractile activity after exposure to a NO donor.

A developing view of the origins of urgency: the importance of animal models

Although caution should be used when applying animal data to human physiology, if care is taken to differentiate between general principles and complications of detail, particular to the species being examined, then experimentation on animal models can reveal basic phenomena in the bladder that offer clues to the origin of urgency. Recent data from the whole isolated bladder of guinea pigs showed unexpected complexities in autonomous activity during the filling phase of the micturition cycle: small, transient increases in intravesical pressure were associated with propagating waves of contractile activity and localized stretches of bladder wall. This complex, coordinated activity suggests that there are mechanisms within the bladder wall devoted specifically to generating phasic activity. Thus, there appear to be two systems controlling detrusor contractions: one associated with overall contractions similar to the micturition contraction and the other generating phasic activity. The mechanisms generating the phasic activity appear to be the point of complex integration of both excitatory and inhibitory inputs. There is evidence that local activity in the bladder wall generates afferent discharge, which probably contributes to bladder sensations. Animal data suggest a novel motor/sensory system incorporating contractile (motor) events, which cause stretches resulting in activation of afferent nerves (sensory). The motor element of this system appears to be controlled in a highly complex fashion such that the amplitude and frequency of the motor activity can be modulated by a variety of inputs. This raises the possibility that the sensitivity of the system informing the central nervous system, and thus awareness of the bladders state during the micturition cycle, can be manipulated, possibly via novel drugs targeted at areas involved in overactive bladder, including urgency incontinence.

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.

A Putative Alternatively Spliced Variant of the P2X1 Purinoreceptor in Human Bladder

Activation of purinergic P2X receptors, putatively P2X1, may be important in the initiation of contraction in human detrusor. Purinergic transmission may be more important in muscle taken from patients with bladder instability. In this study the presence of the P2X1 receptor subtype was confirmed using RT‐PCR. In addition, the results indicate, at the mRNA level, the presence of a splice variant of P2X1 that is lacking part of the second transmembrane domain. It is therefore possible that human bladder expresses multiple isoforms of the P2X1 receptor which may be potential sites for modifying or regulating putative purinergic activation of the human bladder.

Bladder volume alters cholinergic responses of the isolated whole mouse bladder

PURPOSEnThe isolated bladder expresses autonomous activity, which may contribute to the generation of lower urinary tract sensation or pathophysiology. We evaluated how the effect of a cholinergic agonist on autonomous activity alters with increasing volume and in the presence of substances known to modulate functional bladder capacity.nnnMATERIALS AND METHODSnThe bladder of 22 adult female C57 black mice were mounted in whole organ tissue baths. Recordings of intravesical pressure were performed under standardized conditions at different bladder volumes.nnnRESULTSnAt low volume the muscarinic agonist arecaidine elicited an initial peak response, which subsided to a sustained steady state pressure. At high volume phasic pressure fluctuations were also apparent. An M2-receptor antagonist caused a significantly greater decrease in peak and steady state responses than in pressure fluctuations. An M3-receptor antagonist decreased all 3 components. Alpha, beta-methylene adenosine triphosphate markedly decreased fluctuations, in contrast to norepinephrine, which eliminated the steady state response while preserving fluctuations.nnnCONCLUSIONSnThe response to cholinergic stimulation of the isolated bladder has 3 components. The initial tonic peak response increases with bladder distention and it is inhibited by M2 and M3 muscarinic receptor antagonists. The tonic steady state response does not vary with bladder volume and it is inhibited by M2 and M3-receptor antagonists, and by beta3-adrenergic receptor agonists. Phasic fluctuations are minimal at low bladder volume, and with alpha, beta-methylene adenosine triphosphate or an M3-receptor antagonist. Thus, the response to cholinergic stimulation varies with bladder volume. It can be differentially modulated by muscarinic antagonists and also by agents acting through nonmuscarinic receptors.