"Focused introspection" during naturally increased diuresis: description and repeatability of a method to study bladder sensation non-invasively.

OBJECTIVE: To present and describe a non-invasive method to study the origin and development of bladder filling sensation and to evaluate the repeatability of the method. METHOD: Eighteen volunteers participated in the study and were given a water loading protocol consisting of 1,000 ml water intake 1 hr before the session and 200 ml every 10 min during the session. Protocol 1: To evaluate diuresis rate, seven participants were asked to void every 15 min and the voided volume was measured. Protocol 2: Eleven volunteers graded bladder sensation on regular time points, on an empty graph with time on the X-axis and intensity of sensation on the Y-axis. The protocol ended at absolute need to void (maximal intensity) and voided volumes were measured. This protocol was conducted three times with a 10 days interval. RESULTS: Protocol 1: The diuresis rate was not different during the sessions and showed no variation over the studied time period (P = 0.2). Protocol 2: For an individual, the diuresis rate was not different between the sessions. The curves in all patients showed a continuously increasing bladder intensity. In seven participants the curve was convex, in the other four, the curve was sigmoidal. For each individual the pattern was constant during the three sessions. CONCLUSION: A strict water loading protocol induces a constant diuresis. This allows individuals to draw an introspection bladder sensation curve with a specific shape, which can be used as a method to study the development of bladder sensation non-invasively.

Modulation of non-voiding activity by the muscarinergic antagonist tolterodine and the ß(3)-adrenoceptor agonist mirabegron in conscious rats with partial outflow obstruction.

UNLABELLED: Experimental urethral obstruction in rats alters micturition patterns with non-voiding activity (NVA) during filling cystometry, showing similarity to that observed in human detrusor overactivity. Several drug classes with therapeutic potential in overactive bladder in humans have been tested in this model in rats, rabbits or guinea pigs, but no detailed analysis of drug effects on cystometric patterns has been published. The present study uses a rat model of overactivity with partial bladder outflow obstruction (BOO) in combination with the procedures to analyse NVA to study the effects of the anticholinergic drug tolterodine and the novel ß(3)-adrenoceptor agonist mirabegron. The current data for the first time show that NVA in rats with BOO is sensitive to both the muscarinergic antagonist tolterodine and the ß(3)-adrenoceptor agonist mirabegron, but with clear differences between the two drugs: during progression of bladder filling, tolterodine affected both the amplitude and frequency of NVA whereas mirabegron affected primarily the frequency. In addition, tolterodine dose-dependently reduced voiding contractions, while mirabegron did not. A model is proposed to account for these observations where both agents act on a 'pacemaker-like' mechanism which is sensitive to cholinergic excitatory and beta-adrenergic inhibitory inputs. Such concepts could provide insights into the nature of overactive bladder and the site of action of key therapeutic drugs. OBJECTIVE: To investigate the hypothesis that tolterodine and the ß(3)-adrenoceptor agonist mirabegron exert their actions on the motor component of the motor/sensory system in the bladder wall: non-voiding activity (NVA). MATERIALS AND METHODS: The present study used standard cystometric techniques and a conscious rat model of partial bladder outflow obstruction (BOO). A single dose of either tolterodine (0.01, 0.1 0.3 or 1.0 mg/kg) or mirabegron (0.03, 0.1, 0.3, 1.0 or 3.0 mg/kg) was given i.v. to each animal. RESULTS: In the dose ranges used, tolterodine reduced the voiding contraction amplitude, whereas mirabegron did not. Non-voiding activity consisted of small (<0.6 mmHg) and large (>0.6 mmHg) transients. As a fill progressed, both tolterodine and mirabegron reduced the cumulative activity of the large non-voiding contractions, but had little effect on the small transients. Tolterodine affected both the amplitude and frequency of NVA, whereas mirabegron affected primarily the frequency. CONCLUSIONS: Non-voiding activity is sensitive to muscarinergic antagonists and ß(3)-adrenoceptor agonists, but there are clear differences between the two drugs. A model is proposed to account for these observations where both agents act on a 'pacemaker-like' mechanism with cholinergic excitatory and adrenergic inhibitory inputs. Such concepts may provide insights into the nature of overactive bladder and the site of action of key therapeutic drugs.

The relationship between prostaglandin E receptor 1 and cyclooxygenase I expression in guinea pig bladder interstitial cells: proposition of a signal propagation system.

PURPOSE: We explored the structural relationship between enzymes producing prostaglandin (cyclooxygenase I) and 1 of the receptor families that respond to prostaglandin (prostaglandin E receptor 1) in the bladder muscle. MATERIALS AND METHODS: Nine male guinea pigs were sacrificed by cervical dislocation. Bladders were removed and fixed in 4% paraformaldehyde in phosphate buffered saline. Frozen sections (10 µm) were cut and stained with antibodies to prostaglandin E receptor type 1, cyclooxygenase I and vimentin. RESULTS: Prostaglandin E receptors 1 was identified on smooth muscle cells, and vimentin positive surface muscle and intramuscular interstitial cells. Muscle staining was less intense than on interstitial cells and had a punctuate appearance. Prostaglandin E receptor 1 expression on interstitial cells was highly localized. Discrete regions of intense staining were noted on interstitial cell processes. Cyclooxygenase I was also expressed in muscle interstitial cells. Cyclooxygenase I positive interstitial cells were more prevalent in the muscle bundles of the inner muscle than in the outer muscle layers. Cyclooxygenase I staining was noted on discrete regions of the cell or cell processes. Double staining with prostaglandin E receptor 1 and cyclooxygenase I suggested that cell regions expressing the former are different from those expressing the latter. CONCLUSIONS: The discovered arrangement of prostaglandin E receptor 1 and cyclooxygenase I may have the potential to facilitate the propagation of signals in the interstitial cell network. Such a signaling system may have a role in coordinating events, as in bladder pathology, facilitating the global coordinated changes associated with bladder wall remodeling.

M3 muscarinic receptor-like immunoreactivity in sham operated and obstructed guinea pig bladders.

PURPOSE: Type 3 muscarinic receptors, which are present in the bladder wall, are important for bladder function. However, their role in the context of the urothelium is not well defined. Understanding the role of type 3 muscarinic receptors has been limited by the lack of specific type 3 muscarinic receptor antibodies. Thus, we identified a specific type 3 muscarinic receptor antibody and investigated the site of type 3 muscarinic receptors in sham operated and obstructed guinea pig bladders. MATERIALS AND METHODS: The specificity of 4 commercially available type 3 muscarinic receptor antibodies was determined. Immunohistochemistry was then done in bladder tissue from sham operated and obstructed guinea pig bladders. RESULTS: One of the 4 antibodies examined had the needed specificity in terms of blocking peptide and Western blot characterization. Using this antibody type 3 muscarinic receptor immunoreactivity was associated with muscle cells, nerves and interstitial cells. Four types of interstitial cells were identified, including suburothelial, lamina propria, surface muscle and intramuscular interstitial cells. In the obstructed model the bladder wall was hypertrophied and there was nerve fiber loss. The number of lamina propria, surface muscle and intramuscular interstitial cells was increased but not the number of suburothelial interstitial cells. Also, surface muscle interstitial cells appeared to form clusters or nodes with type 3 muscarinic receptor immunoreactivity. CONCLUSIONS: Nerve loss and the up-regulation of interstitial cells with type 3 muscarinic receptor immunoreactivity may underlie major functional changes in the pathological bladder. This indicates that type 3 muscarinic receptor specific anticholinergic drugs may affect not only the detrusor muscle, as previously thought, but also interstitial cells and nerve fibers.

Nonvoiding activity of the guinea pig bladder.

PURPOSE: Detrusor nonvoiding contractions occur in up to 70% of healthy individuals. These contractions increase in those with pathological detrusor function and may be associated with afferent activity. We examined nonvoiding contractions in the urethane anesthetized guinea pig bladder and studied the effect of filling rate and intravesical volume. MATERIALS AND METHODS: A total of 14 guinea pigs were anesthetized and underwent bladder catheterization at the dome. In 6 guinea pigs bladder infusion was continuous and 2 physiological filling rates were used, including 25 (0.75 HD) and 50 µl per minute (1.5 HD). In another 8 guinea pigs isovolumetric cystometry was done by filling the bladder incrementally and recording at low, medium and high intravesical volume. RESULTS: Nonvoiding contractions were apparent in all animals. Contractions increased in frequency and amplitude as the bladder filled. Different phases were identified. Immediately after a void no nonvoiding contractions were observed, followed by continuous activity, first with small contractions, and later with small and large contractions. Small nonvoiding contractions showed a phasic pattern in frequency while the frequency of large nonvoiding contractions slowly increased or remained stable. The frequency and amplitude of nonvoiding contractions were higher at a faster filling rate and a higher intravesical volume. CONCLUSIONS: Nonvoiding contractions are present in the anesthetized guinea pig. Under normal physiological conditions they increase in amplitude and frequency with the increase in the filling rate and in intravesical volume. Small and large nonvoiding contractions differ in frequency pattern and occur at different bladder filling periods. This may illustrate different afferents functioning during bladder filling, which could be important for understanding bladder pathology.

Structural and functional relations between the connective tissue and epithelium of enamel organ and their role during enamel maturation.

The morphological and possible functional interactions between the connective tissue and enamel organ cells were examined during the maturation phase of enamel formation, using immunohistochemical techniques. Decalcified mandibular sections (10 µm) including incisors were used from Wistar rats ages 10-12 weeks. Sections were incubated with one or two primary antibodies targeting cell cytoskeleton (vimentin, α-actin, α-tubulin), dendritic marker (OX6), gap junctions (cx-43), enzymes (nitric-oxide synthase (nos1) and cyclooxygenase (cox1)), and the ion transporters (Na+/H+ exchanger (NHE1) and Na+/Ca2+ exchanger (NCX)) for 24 h, before incubation with the appropriate conjugated fluorescent secondary antibodies. Sections were examined by fluorescence microscopy. Haematoxylin-eosin slides were also employed. Cellular heterogeneity and morphological modulations were identified within enamel organ cells and connective tissue covering suggesting complex cellular interactions and indicating a new functional concept and possible complementary role during enamel maturation. Also, some ion transportation activity, and nos1 and cox1 signalling pathways have been identified, indicating intercellular communication between these regions. A hypothesis is suggested, to explain the morphological modulation of ameloblasts and papillary cells during enamel maturation which functions to increase the transporting membrane surface area to accomplish faster and bulker ion transportation to achieve controlled pH and to direct Ca2+ towards enamel.

Prostaglandin receptor EP1 and EP2 site in guinea pig bladder urothelium and lamina propria.

PURPOSE: Urothelium has 2 main functions. It is a barrier to urine and has a sensory role. In response to stretch urothelium releases various substances that modulate afferent nerve activity. Recent data on the localization of cyclooxygenase type 1, the enzyme responsible for prostaglandin production, suggests that prostaglandin may have complex local action. MATERIALS AND METHODS: The bladders of 7 guinea pigs were stained for prostaglandin receptors type 1 and 2, and costained for vimentin and cyclooxygenase I. RESULTS: Prostaglandin receptor type 1 staining was seen in urothelial cells and in the suburothelium. Urothelial staining, which was often punctuate and weak, was detected in all urothelial cell layers, including suburothelial cells. In contrast, strong prostaglandin receptor type 2 staining was seen in the urothelium and in suburothelial cells. Cyclooxygenase I was absent in interstitial cells and umbrella cells with the highest concentration in the basal cell layer. CONCLUSIONS: Interstitial cells express prostaglandin receptor types 1 and 2, indicating that they can respond to prostaglandin. Umbrella cells do not express cyclooxygenase I. Cyclooxygenase I was present in basal urothelial cells, making them a possible site of prostaglandin synthesis. Thus, prostaglandin produced by urothelium may target prostaglandin receptor types 1 and 2 in the urothelium and suburothelium. Therefore prostaglandin is hypothesized to have a role in signal regulation in the bladder wall.

Stretch independent regulation of prostaglandin E(2) production within the isolated guinea-pig lamina propria.

OBJECTIVE: To use an isolated preparation of the guinea-pig bladder lamina propria (LP) to investigate the effects of adenosine tri-phosphate (ATP) and nitric oxide (NO) on the release of prostaglandin E(2) (PGE(2)). MATERIALS AND METHODS: The bladders of female guinea-pigs (200-400 g) were isolated and opened to expose the urothelial surface. The LP was dissected free of the underlying detrusor muscle and cut into strips from the dome to base. Strips were then incubated in Krebs buffer at 37 degrees C. Each tissue piece was then exposed to the stable ATP analogue, BzATP, and a NO donor, diethylamine-NONOate (DEANO), and the effect on PGE(2) output into the supernatant determined using the Parameter(TM) PGE(2) enzyme immunoassay kit (R & D Systems, Abingdon, UK). Experiments were repeated in the presence of purinergic receptor and cyclooxygenase (COX) enzymes, COX I and COX II, antagonists. The cellular location of COX I, COX II and neuronal NO synthase (nNOS) within the bladder LP was also determined by immunohistochemistry. RESULTS: PGE(2) production was significantly increased by BzATP. Antagonist studies showed the purinergic stimulation involved both P(2)X and P(2)Y receptors. The BzATP response was inhibited by the COX inhibitor indomethacin (COX I >COX II) but not by DUP 697 (COX II >COX I). Thus, BzATP stimulation occurs because of COX I stimulation. NO had no effect on PGE(2) production over the initial 10 min of an exposure. However, PGE(2) output was increased 100 min after exposure to the NO donor. In the presence of NO, the BzATP stimulation was abolished. Immunohistochemistry was used to confirm the location of COX I to the basal and inner intermediate urothelial layers and to cells within the diffuse layer of LP interstitial cells. In addition, nNOS was also located in the basal urothelial layers whilst COX II was found in the interstitial cell layers. CONCLUSIONS: There is complex interaction between ATP and NO to modulate PGE(2) release from the bladder LP in the un-stretched preparation. Such interactions suggest a complex interrelationship of signals derived from this region of the bladder wall. The importance of these interactions in relation to the physiology of the LP remains to be determined.

On the origins of the sensory output from the bladder: the concept of afferent noise.

For many people a recurrent strong desire to void, sometimes with incontinence, diminishes their quality of life. At present there are few insights into what underlies these problems. The condition is described as the 'overactive bladder symptom complex' but this definition is proving to be unhelpful. It focuses on overt bladder contractions rather than the main problem, which is altered and heightened sensation. Also, current approaches that describe bladder sensations as episodic and leading to voiding as 'first and second sensation to void' might also be misleading if they are taken too literally and used to suggest mechanisms. Current research is beginning to focus on the mechanisms that generate afferent information from the bladder and how it can become altered. As these views develop it is crucial that we appreciate the diversity of the bladder afferent system and distinguish between afferent and sensory information; in this review we explore this underlying complexity. The central nervous system (CNS) receives vast amounts of information from the bladder, which arises from different locations, uses different fibre types and involves different methods. The CNS is continually being bombarded with 'afferent noise'. The challenge now is to understand the nature and components of this 'afferent noise' and which components are essential to sensation. The emerging picture is complex, but this complexity must not be negated or oversimplified. It must be embraced and incorporated it into thinking when designing experiments, analysing data, diagnosing patients and evaluating treatment.

Ubiquitin hydrolase (protein gene product 9.5) in the obstructed bladder: evidence for tissue remodelling involving a subset of interstitial cells.

OBJECTIVE: To examine the expression of ubiquitin hydrolase (UH), an enzyme which is part of the ubiquitin-proteasome system involved in the regulation of cell growth and differentiation, to gain an insight into the cell types and processes underlying the tissue remodelling that occur after bladder neck damage. MATERIALS AND METHODS: Three groups of male guinea pigs were used, comprising controls (not operated, four), sham (five) and obstructed (six). The bladder outlet was obstructed by implanting a silver ring around the urethra, which was left in situ for 2-4 weeks. Sham-operated guinea pigs had the same operative procedure but no ring was implanted. The bladders were removed and incubated in Krebs' solution at 36 degrees C, gassed with 95% O2 and 5% CO2, Tissues were then fixed in 4% depolymerized paraformaldehyde and processed for immunohistochemistry. We used antibodies raised against UH, cyclooxygenase type I and vimentin. Specific antibody binding was visualized using the appropriate secondary antibodies. RESULTS: Staining with an antibody to UH showed the presence of both sensory and motor nerves in control, sham and obstructed bladders. In the control bladders this was the predominant staining pattern. In the sham and obstructed bladders UH staining revealed additional positive cell types; cells associated with the outermost layers of the urothelium (the umbrella cells), in the lamina propria (the lamina propria interstitial cells (lp-ICs), on the surface of the muscle bundles (surface muscle, sm-ICs) and on the serosal surface (muscle coat, mc-ICs). All ICs stained with vimentin. The ICs within the muscle bundles (intramuscular, im-ICs) did not stain with UH. The number and density of the UH-positive cells was greater in the obstructed than in the sham bladders, suggesting a change in relation to the severity of damage to the bladder neck. CONCLUSION: The expression of UH implies the re-targeting of proteins marked for degradation in the proteasome. Increased expression of UH in the lp-ICs and sm-ICs shows that these cells are active in the early and late stages of the tissue remodelling resulting from obstruction. These results show a further subset of ICs that might be involved in the increased deposition of extracellular material and tissue remodelling.

M(3) muscarinic receptor expression on suburothelial interstitial cells.

OBJECTIVE: To identify the cells expressing the M(3) muscarinic receptor subtype in the lamina propria of the bladder. MATERIALS AND METHODS: The bladders from five normal guinea pigs were isolated and fixed in 4% paraformaldehyde. Tissues sections (10 microm) were then cut and stained with antibodies to the type 3 muscarinic receptor (M(3)), the interstitial cell marker vimentin and the nonspecific nerve marker PGP 9.5. The specificity of the antibody to the M(3) receptor was established using the complementary blocking peptide and Western blot analysis of human embryonic kidney (HEK) cells transfected to express the M(3) receptor protein. RESULTS The M(3) antibody pre-incubated with its blocking peptide showed no immunohistochemical staining. Investigating this antibody using HEK cells transfected to express the M(3) receptor protein and control HEK cells showed a single band in the transfected cells and no band in the control cells. M(3) receptor immunoreactivity (M(3)-IR) was detected primarily on a dense network of vimentin-positive (vim(+)) cells lying immediately below the urothelium, i.e. the suburothelial interstitial cells (Su-ICs). The M(3)-IR was punctate and appeared to be located on the cell surface. The diffuse network of cells in the remaining regions of the lamina propria showed no M(3)-IR. Few nerve fibres were associated with the M(3)-IR Su-ICs. The M(3)-IR Su-ICs were most numerous and prominent in the lateral wall. The number of M(3)-IR/vim(+) cells diminished towards the bladder base and were absent in the bladder urethral junction. In the base and urethral junction there were vim(+) cells that were not M(3)-IR. A population of umbrella cells in the lateral wall also showed weak punctate M(3)-IR. CONCLUSIONS Using a well-characterized M(3) antibody, these results show for the first time that the M(3) muscarinic receptor in the lamina propria is located specifically on the Su-ICs. The physiological role of these cells is unknown and consequently the significance of what appears to be a cholinergic signalling system is unclear. Previously published data showed that these cells respond to nitric oxide and atrial natriuretic peptide with an increase in cGMP and possibly prostaglandin. All of these observations, taken together, suggest that the Su-ICs receive multiple inputs and that they must be part of a complex signalling system in this region of the bladder wall.

Volume-induced responses in the isolated bladder: evidence for excitatory and inhibitory elements.

OBJECTIVE: To quantify changes in autonomous activity in response to alterations in intravesical volume, to explore the possible underlying regulatory mechanisms. MATERIALS AND METHODS: Experiments were conducted using whole isolated bladders from six female guinea pigs (280-400 g). A cannula was inserted into the urethra to monitor intravesical pressure and the bladder was suspended in a heated chamber containing carboxygenated physiological solution at 33-36 degrees C. All drugs were added to the solution on the ablumenal bladder surface. RESULTS: An increase in intravesical volume followed by a rapid reduction lead to a complex series of activity comprising of several distinct phases. After a volume increase there was an initial 'burst' of frequency which gradually declined to a 'steady state'. After a volume reduction there was a period of quiescence with spontaneous activity gradually returning to levels seen before the increase, termed the 'inhibitory phase'. The frequency of transient contractions, both immediately after a volume increase and at steady state, increased both with increasing intravesical volume and dose of arecaidine. The length of the inhibitory phase increased both with the duration and magnitude of volume increase. However, the inhibitory phase was not entirely dependent n the magnitude of volume change, as the inhibitory phase was shorter when the volume was not returned to baseline levels. Increasing doses of arecaidine shortened the inhibitory phase. CONCLUSIONS: These observations suggest that the regulation of volume-induced spontaneous activity relies on complex excitatory and inhibitory inputs. The rapid burst of activity resulting from a rise in volume suggests the presence of a rapidly adapting mechanism. Rapid reduction in intravesical volume leads to a quiescent period, i.e. the inhibitory phase. This is related to both the duration of intravesical volume increase and its magnitude. However, similar volume changes are more effective when the volume is reduced back to baseline, as opposed to the bladder being incompletely emptied. Furthermore, the frequency of transient contractions remained constant once a steady state was reached, with no evidence of inhibition before volume reduction. This suggests that mechanisms involved in the generation of the inhibitory phase initiated during bladder filling require >30 s to have a significant effect, but depend on a reduction in volume to be triggered, with the response dependent on the volume reduced. The mechanisms involved in generating and modulating the inhibitory phase seem to be regulated by a strong cholinergic input but the exact nature of these mechanisms is unknown. The potential importance of these results in terms of the general physiology and pharmacology of the bladder is discussed.

Regional differences in sensory innervation and suburothelial interstitial cells in the bladder neck and urethra.

OBJECTIVE: To identify and characterize possible structural specialisations in the wall of the lower urinary tract (LUT) in the region of the bladder urethral junction (BUJ), with the specific objective of identifying regional variations in sensory nerve fibres and interstitial cells (ICs). MATERIALS AND METHODS: The bladder base and urethra was removed from five male guinea pigs killed by cervical dislocation. Tissue pieces were incubated in Krebs' solution at 36 degrees C, gassed with 95% O(2) and 5% CO(2), fixed in 4% paraformaldehyde and processed for immunohistochemistry. The nonspecific marker vimentin and the general neuronal marker protein gene product (PGP) 9.5 were used to identify ICs and nerve fibres, respectively. Specific antibody binding was visualized using the appropriate secondary antibodies. RESULTS: The wall of the LUT in the region immediately between the bladder base and the urethra, the BUJ, differed in its cellular composition relative to the adjacent areas. PGP-positive (PGP(+)) nerve fibres, presumptive afferent fibres, lay within the urothelium running between the epithelial cells. There were two general nerve patterns: branching fibres with no varicosities, and complex fibres with varicosities. Fibre collaterals with varicosities exited the urothelium and occupied the space under the urothelium adjacent to the layer of suburothelial ICs. The latter, lamina propria and around the muscle bundles were identified using vimentin (vim(+)). In the base a few vim(+) cells were also PGP(+). In the region of the BUJ there was a decrease in the amount of smooth muscle. In this region, below the lamina propria, there was an area densely populated with vim(+)/PGP(+) ICs. Nerve fibres ran between the cells in this region. CONCLUSION: These structural specialisations within the urothelium and deeper layers of the BUJ suggest that they might be associated with specific functions. The localized highly branched network of the putative afferent nerves suggests the presence of a local axonal reflexes involving possible cross-talk between the urothelium and suburothelial layer. The function of the specialized region of ICs is not known and must await further information on the functional properties of this novel cell type. These observations show further the cellular heterogeneity of the cells in the LUT and the complexity of the structures. One of the major current challenges in functional urology is to understand the relationships between these novel structures and overall bladder and urethral function.

The actions of prolonged exposure to cholinergic agonists on isolated bladder strips from the rat.

The present study was done to explore the cholinergic systems operating in the wall of the isolated rat bladder. In a first set of experiments, bladder strips in vitro were subjected to cumulative concentration-response curve (CRC) to non-selective muscarine agonist carbachol or the partially M2>M3 selective agonist arecaidine to establish optimal concentration to be used thereafter. In a second set of experiments, the effects of drugs (solifenacin, isoproterenol, and mirabegron) were tested on urinary bladder contraction induced by the non-selective muscarinergic agonist carbachol. For both agonists, the contractile responses are qualitatively similar: an initial transient rise in tension followed by complex bursts of high-frequency small 'micro'-contractions superposed on a tonic contraction, with immediate transient 'rebound' contraction after the agonist is washed from the preparation. This rebound contraction is greater with carbachol than arecaidine. Components of the responses to cholinergic stimulation, notably the micro-contractions, were found to be differently stimulated and inhibited by the M3>M2 selective antagonist solifenacin and by the ß-adrenoceptor agonists isoprenaline and mirabegron. A physiological role for the muscarinic dependent phasic contractions and the micro-anatomical elements that might be involved are not known but may be related to non-voiding activity observed during filling cystometry in conscious animals related to afferent discharge and possibly sensation. Furthermore, suggestions for the potential impact of these findings and design of further studies in relation to bladder physiology, pharmacology, and pathology are discussed.

Smooth muscle alpha-actin expression in endothelial cells derived from CD34+ human cord blood cells.

Human fetal cord blood contains subsets of mononuclear cells with the potential to form both hematological and endothelial cells. Vascular progenitor cells, which can produce all three elements of mature blood vessels, including smooth muscle, have been identified in animals. We hypothesized that similar multipotential progenitor cells exist in humans and used the expression of alpha-smooth muscle actin (alpha-SMA) to identify such cells in fetal cord blood. Mononuclear cell preparations were isolated from human umbilical cord blood and CD34(+) and CD133(+) cells obtained by magnetic bead separation. Isolated cells were cultured on fibronectin-coated dishes with medium containing vascular endothelial growth factor, basic fibroblast growth factor, and insulin-like growth factor. mRNA was extracted, and the expression of alpha-SMA and a number of endothelial cell markers (VEGFR-2, vWF, eNOS, VE-Cadhein, PECAM-1 and Tie-2) was determined by reverse transcriptase-PCR techniques. Human umbilical vein endothelial cells (HUVECs) were used as positive controls. Freshly isolated CD34(+) and CD133(+) cells expressed all endothelial cell markers, but did not express alpha-SMA. HUVECs expressed alpha-SMA. Following 4 weeks of culture, CD34(+) isolates produced morphologically endothelial-like cells that expressed both endothelial cell markers and alpha-SMA. CD133(+) cells failed to produce morphological endothelial-like cells but expressed a range of endothelial markers. However, they did not express alpha-SMA. Following culture in an endothelial cell-promoting environment, CD34(+), but not CD133(+), isolates produced endothelial-like cells that expressed alpha-SMA. Human fetal cord blood contains a population of cells that may differentiate toward both an endothelial and a smooth muscle phenotype in culture.

The concept of peripheral modulation of bladder sensation.

It is recognized that, as the bladder fills, there is a corresponding increase in sensation. This awareness of the volume in the bladder is then used in a complex decision making process to determine if there is a need to void. It is also part of everyday experience that, when the bladder is full and sensations strong, these sensations can be suppressed and the desire to void postponed. The obvious explanation for such altered perceptions is that they occur centrally. However, this may not be the only mechanism. There are data to suggest that descending neural influences and local factors might regulate the sensitivity of the systems within the bladder wall generating afferent activity. Specifically, evidence is accumulating to suggest that the motor-sensory system within the bladder wall is influenced in this way. The motor-sensory system, first described over 100 years ago, appears to be a key component in the afferent outflow, the afferent "noise," generated within the bladder wall. However, the presence and possible importance of this complex system in the generation of bladder sensation has been overlooked in recent years. As the bladder fills the motor activity increases, driven by cholinergic inputs and modulated, possibly, by sympathetic inputs. In this way information on bladder volume can be transmitted to the CNS. It can be argued that the ability to alter the sensitivity of the mechanisms generating the motor component of this motor-sensory system represents a possible indirect way to influence afferent activity and so the perception of bladder volume centrally. Furthermore, it is emerging that the apparent modulation of sensation by drugs to alleviate the symptoms of overactive bladder (OAB), the anti-cholinergics and the new generation of drugs the ß 3 sympathomimetics, may be the result of their ability to modulate the motor component of the motor sensory system. The possibility of controlling sensation, physiologically and pharmacologically, by influencing afferent firing at its point of origin is a "new" concept in bladder physiology. It is one that deserves careful consideration as it might have wider implications for our understanding of bladder pathology and in the development of new therapeutic drugs. In this overview, evidence for the concept peripheral modulation of bladder afferent outflow is explored.

Cholinergic activation of phasic activity in the isolated bladder: possible evidence for M3- and M2-dependent components of a motor/sensory system.

OBJECTIVES: To analyse pressure changes induced by muscarinic agonists on the isolated bladder in order to examine whether there are different responses representing different components of a motor/sensory system within the bladder wall. MATERIALS AND METHODS: Whole isolated bladders from 19 female guinea-pigs (280-400 g) were used. A cannula was inserted into the urethra to monitor intravesical pressure and the bladder was suspended in a heated chamber containing carboxygenated physiological solution at 33-36 degrees C. Initially, the responses to the cholinergic agonists, arecaidine but-2-ynyl ester tosylate and carbachol were assessed. Then, in an attempt to identify the muscarinic receptor subtypes involved, the effects of selective muscarinic antagonists on the arecaidine-induced bladder responses were assessed. The antagonists used were the relatively M(3)-selective 4-diphenylacetoxy-N-methylpiperidine methobromide (4-DAMP) and darifenicin, and relatively M(2)-selective AFDX-116. All drugs were added to the solution bathing the ablumenal surface of the bladder. RESULTS: The whole bladders exposed to cholinergic agonists respond with complex changes in intravesical pressure. Immediately after application of the agonist there was a burst of high frequency transient contractions. During continued application of agonist the frequency of the transients decreased and their amplitude increased. Thus, there appear to be two components to the response: an initial fast phase and a later slow component. The maximum frequency of the initial burst increased with increasing concentrations of agonist. By contrast, the frequency of the transients in the steady state showed little dependence on agonist concentration. There were quantitative differences between the responses to arecaidine and carbachol. Arecaidine was less effective in generating the initial burst of high-frequency activity and the transients were significantly larger. At low dose, arecaidine was more effective in producing the large transients in the steady state. Pre-exposure of the bladder to 4-DAMP (0.1-10 nM) or darifenicin (0.1-10 nM) significantly reduced the frequency of the initial burst of activity; 0.3 nM 4-DAMP reduced the frequency by half. In this concentration range, 4-DAMP reduced the amplitude of the initial transients but did not affect the frequency of the transients in the steady state. There were similar results with darifenicin. However, darifenicin was less effective in reducing the amplitude of the initial transients. By contrast, ADFX-116 had little effect on the frequency of the initial transients but did reduce amplitude; 300 nM AFDX-116 was needed to reduce the frequency of the initial burst by half. CONCLUSIONS: This analysis suggests that there are different but interrelated mechanisms in the isolated bladder contributing to complex contractile activity. Three components can be identified: a mechanism operating during voiding to produce a global contraction of the whole bladder and two mechanisms, pacemaker and conductive, involved in generating and propagating local contractions in the bladder wall. The pacemaker component is more sensitive to darifenicin and 4-DAMP than to AFDX-116 suggesting that the underlying processes rely predominantly on M(3) receptors and less so on M(2) (M(3) > M(2)). The phasic activity in the later stages is less affected by M(3) antagonists and might therefore involve predominantly M(2) receptors (M(2) > M(3)). The potential importance of these results in terms of the general physiology and pharmacology of the bladder is discussed.

The effects of exogenous prostaglandins and the identification of constitutive cyclooxygenase I and II immunoreactivity in the normal guinea pig bladder.

OBJECTIVES: To establish the functional consequences of exposing the isolated whole bladder preparation to exogenous prostaglandins (PGE(1), PGE(2), PGF(2alpha)) and to determine which cells express cyclooxygenase (COX) types I and II, to generate PG to effect these changes in vivo. MATERIALS AND METHODS: Fifteen female guinea pigs (270-350 g) were used, i.e. seven for structural studies and eight for physiological measurement. For the structural study pieces of the lateral wall were incubated separately in Krebs' solution at 36 degrees C, gassed with 95% O(2) and 5% CO(2) with 1 mm isobutyl-methyl-xanthene. Individual pieces were then exposed to 100 microm of the nitric oxide (NO) donor NONOate for 10 min; control tissues remained in Krebs' solution. Tissues were then fixed in 4% paraformaldehyde. For the physiological experiments bladders were isolated and a cannula inserted into the urethra to monitor intravesical pressure. The bladders were suspended in a chamber containing carboxygenated physiological solution at 33-36 degrees C. All drugs were added to the abluminal bladder surface. RESULTS: In the resting bladder there were small spontaneous transient rises in pressure, i.e. autonomous activity. Exposure to PGE(2) (3-300 nM) resulted in an increase in basal pressure on which were superimposed autonomous activity, which was increased both in amplitude and frequency. The changes in the amplitude and frequency depended on the concentration of PGE(2). After a brief exposure (240 s) to PGE(2) the augmentation of the autonomous activity continued for >60 min despite regular washing. The responses were similar with PGE(1) but the responses to PGF(2alpha) and arachidonic acid were reduced. The augmented activity was reduced by the EP1/EP2 receptor blocking agent AH6809 (10 microm). Using an antibody to the 70 kDa constitutive form (COX I), COX I immunoreactivity (COX I-IR) was located in cells in the basal urothelium, in lamina propria and cells on the surface of the inner muscle bundles. There were few COX I-IR cells associated with the outer muscle bundles. The COX I-IR cells lying within the lamina propria were distinct from the suburothelial cells which respond to NO with an increase in cGMP. The lamina propria COX I-IR cells appeared to form a network surrounding muscle trabeculae within the inner muscle layer. COX II-IR was associated with the nuclei of cells in the urothelium, lamina propria and muscle. CONCLUSIONS: These data show that PGs regulate autonomous activity. Potential sources of endogenous PG were identified. It is unclear how the PGs produced by these cells alter autonomous activity. There might be a direct activation of the muscle by PGs released by the network of superficial muscle interstitial cells, or PG released from the urothelium might influence phasic contractile activity via networks of COX I-IR interstitial cells. The possible roles and importance of this mechanism for bladder physiology and pathology are discussed.

Damage to the bladder neck alters autonomous activity and its sensitivity to cholinergic agonists.

OBJECTIVE: To identify and describe changes to the motor component of the motor/sensory system, which contributes to sensation during the filling phase of the micturition cycle, as a result of surgically induced bladder pathology, i.e. damage to the bladder neck and outlet obstruction. MATERIALS AND METHODS: Adult male guinea pigs (294-454 g) were assigned initially into three groups: (i) normal guinea pigs with no surgical intervention (control, seven); (ii) guinea pigs which, with full surgical anaesthesia, had a silver ring implanted around the bladder neck (obstructed, 13); and (iii) guinea pigs operated to expose the bladder neck but with no implantation of a ring (sham, six). At 2-4 weeks after surgery the bladders were isolated, weighed and the pressure recordings used to identify autonomous activity. RESULTS: The bladder weights in all operated groups, including the sham, were greater than controls. Bladder weights in the obstructed guinea pigs varied considerably, reflecting the degree of pathological change. Consequently, bladders from this group were divided into those with high (OBH) and those with low bladder weight (OBL). The mean (sd) amplitudes of the autonomous contractions were 1.1 (0.1), 10.8 (1.8), 11.4 (2.5) and 17.1 (4.0) cmH(2)O in control, sham, OBL and OBH bladders, respectively, indicating a progressive alteration in function with the pathology. The changes in the sham group suggested that the pathological changes were not the result of obstruction but damage to the bladder neck, the implantation of the silver rings exacerbating the damage. There were episodes of rapid phasic activity (bursts) in 10 of 13 of the ring-implanted bladders, and in two of six in the sham group, but never in controls. Neither the autonomous activity nor the bursts were affected by tetrodotoxin (1 microm) or atropine (3 microm) but they were abolished by noradrenaline (3 microm). In control bladders, adding the muscarinic agonist arecaidine produced a transient acceleration of phasic activity and increased the amplitude of the contractions. There was a similar acceleration of activity in all the operated groups but the concentrations needed to achieve an increase in frequency were significantly lower, the relative sensitivity to arecaidine being OBH >/= OBL > sham > control. CONCLUSION: The mechanism involved in controlling the frequency of the motor component of the motor/sensory system, the 'pacemaker', appears to become progressively 'supersensitive' to cholinergic stimulation with the development of pathology. These observations are discussed in relation to the motor/sensory system and the origins of sensation in the bladder. The argument is proposed that damage to the bladder neck, not obstruction per se, results in altered nonmicturition activity which contributes to increased afferent output. In turn this contributes to the increased sensations of urge associated with bladder dysfunction. The cholinergic regulation of this altered 'pacemaker' might be the target for one of the therapeutic actions of anticholinergic drugs.

Interstitial cells and cholinergic signalling in the outer muscle layers of the guinea-pig bladder.

OBJECTIVES: To explore the relationship between cholinergic mechanisms and interstitial cells (ICs) in the outer muscle layer of the bladder. MATERIALS AND METHODS: In bladder tissue from male guinea-pigs, ICs were identified by their response to nitric oxide (NO) with a rise in cGMP. Sections of the lateral bladder wall were incubated in Krebs' solution containing 1 mm of the nonspecific phosphodiesterase inhibitor isobutyl-methyl-xanthene. Tissues were then exposed to 100 microm of the NO donor NONOate for 10 min, control tissues remained in Krebs' solution. Tissues were then processed for immunohistochemistry for cGMP, choline acetyltransferase (ChAT), neurofilament protein, and the nonspecific neuronal marker protein gene product (PGP) 9.5. RESULTS: cGMP-positive ICs were found mainly in the outer muscle layers of the bladder wall. Three types were identified based on location; on the outer surface of the bladder wall, on the surface of the muscle bundles, and within the muscle bundles. Some of the intramuscular ICs stained for ChAT, but they did not stain with PGP 9.5. Nerve fibres were seen in close contact with the ChAT-positive intramuscular ICs, and these nerves expressed ChAT and neurofilament protein. CONCLUSIONS: A subpopulation of intramuscular ICs can synthesise acetylcholine, and might release acetylcholine onto the underlying muscle. These cells are in close contact with nerves, suggesting that they might be activated by neural inputs. Thus there may be a system in the detrusor involving cholinergic nerves acting on ICs which can activate the smooth muscle via a complex cholinergic input.