Increased gap junction expression in lamina propria myofibroblasts and urothelial cells may be involved in detrusor overactivity, leading to incontinence. Immunohistochemistry was used to compare connexin (Cx) 26, 43, and 45 expression in the bladders of neonatal, adult, and spinal cord-transected rats, while optical imaging was used to map the spread of spontaneous activity and the effects of gap junction blockade. Female adult Sprague-Dawley rats were deeply anesthetized, a laminectomy was performed, and the spinal cord was transected (T8/T9). After 14 days, their bladders and those of age-matched adults (4 mo old) and neonates (7-21 day old) were excised and studied immunohistochemically using frozen sections or optically using whole bladders stained with voltage- and Ca(2+)-sensitive dyes. The expression of Cx26 was localized to the urothelium, Cx43 to the lamina propria myofibroblasts, and Cx45 to the detrusor smooth muscle. While the expression of Cx45 was comparable in all bladders, the expression of Cx43 and Cx26 was increased in neonate and transected animals. In the bladders of adults, spontaneous activity was initiated at multiple sites, resulting in a lack of coordination. Alternatively, in neonate and transected animals spontaneous activity was initiated at a focal site near the dome and spread in a coordinated fashion throughout the bladder. Gap junction blockade (18beta-glycyrrhetinic acid, 1 microM) abolished this coordinated activity but had no effect on the uncoordinated activity in adult bladders. These data suggest that coordinated spontaneous activity requires gap junction upregulation in urothelial cells and lamina propria myofibroblasts.

This study examined the origin of spontaneous activity in neonatal and adult rat bladders and the effect of stretch and muscarinic agonists and antagonists on spontaneous activity. Rats were anesthetized and their bladders were excised, cannulated, and loaded with voltage- and Ca(2+)-sensitive dyes. Intracellular Ca(2+) and membrane potential transients were mapped using photodiode arrays in whole bladders, bladder sheets, or cross-section preparations at 37 degrees C. Intravesical pressure was recorded from whole bladders. In neonatal bladders and sheets, spontaneous Ca(2+) and electrical signals arose at a site near the dome and spread in a coordinated manner throughout the bladder with different dome-to-neck conduction velocities (Ca(2+): 3.7 +/- 0.4 mm/s; membrane potential: 46.2 +/- 3.1 mm/s). In whole bladders, optical signals were associated with spontaneous contractions (10-20 cmH(2)O). By contrast, in adult bladders spontaneous Ca(2+) and electrical activity was uncoordinated, originating at multiple sites and was associated with smaller (2-5 cmH(2)O) contractions. Spontaneous contractions and optical signals were insensitive to tetrodotoxin (2 muM) but were blocked by nifedipine (10 muM). Stretch or low carbachol concentrations (50 nM) applied to neonatal whole bladders enhanced the amplitude (to 20-35 cmH(2)O) of spontaneous activity, which was blocked by atropine. Bladder cross sections revealed that Ca(2+) and membrane potential transients produced by stretch or carbachol began near the urothelial-suburothelial interface and then spread to the detrusor. In conclusion, spontaneous activity in neonatal bladders, unlike activity in adult bladders, is highly organized, originating in the urothelium-suburothelium near the dome. Activity is enhanced by stretch or carbachol and this enhancement is blocked by atropine. It is hypothesized that acetylcholine is released from the urothelium during bladder filling to enhance spontaneous activity.

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

The isolated bladder shows autonomous micromotions, which increase with bladder distension, generate sensory nerve activity, and are altered in models of urinary dysfunction. Intravesical pressure resulting from autonomous activity putatively reflects three key variables; the extent of micromotion initiation, distances over which micromotions propagate, and overall bladder tone. In vivo, these variables are subordinate to the efferent drive of the central nervous system. In the micturition cycle storage phase, efferent inhibition keeps autonomous activity generally at a low level, where it may signal “state of fullness” while maintaining compliance. In the voiding phase, mass efferent excitation elicits generalized contraction (global motility initiation). In lower urinary tract dysfunction, efferent control of the bladder can be impaired, for example due to peripheral “patchy” denervation. In this case, loss of efferent inhibition may enable unregulated micromotility, and afferent stimulation, predisposing to urinary urgency. If denervation is relatively slight, the detrimental impact on voiding may be low, as the adjacent innervated areas may be able to initiate micromotility synchronous with the efferent nerve drive, so that even denervated areas can contribute to the voiding contraction. This would become increasingly inefficient the more severe the denervation, such that ability of triggered micromotility to propagate sufficiently to engage the denervated areas in voiding declines, so the voiding contraction increasingly develops the characteristics of underactivity. In summary, reduced peripheral coverage by the dual efferent innervation (inhibitory and excitatory) impairs regulation of micromotility initiation and propagation, potentially allowing emergence of overactive bladder and, with progression, detrusor underactivity.

We examined the modulation of intrinsic (i.e., spontaneous) detrusor contractions by the urothelium and the lamina propria through optical mapping approaches. Normal adult and spinal cord-transected (SCT) rat bladders were stained with Ca2+- and voltage-sensitive dyes, and optical activity generated from intrinsic contractions was mapped from the mucosal surface of whole bladder sheets. Both normal adult and SCT rat bladders displayed intrinsic contractions, where normal bladders showed low-amplitude, high-frequency contractions with disorganized patterns of activity. In contrast, in the SCT animals there were high-amplitude, low-frequency contractions that displayed an organized spread of membrane potential and intracellular Ca2+. The difference in contractile activity was mirrored in the Ca2+ and membrane potential maps of bladder sheets. Normal bladders showed multiple initiation sites across the mucosal surface, whereas SCT bladders showed only one or two fixed initiation sites localized to the dome. The magnitude of intrinsic contractions could be enhanced by stretch or low-dose arecaidine (50 nM), a muscarinic-specific agonist. Partial removal of the mucosa decreased the amplitude of the intrinsic contractions and decreased the response to stretch or arecaidine. Optical mapping of mucosa-denuded sheets, where enhanced spontaneous activity was abolished, or application of 1 microM nifedipine to remove smooth muscle signals, but not the mucosal signals, shows that intrinsic activity in pathological bladders is driven by the mucosal layer. In summary, we suggest an urotheliogenic origin for intrinsic activity, where structures within the mucosal layer organize and thereby enhance intrinsic detrusor contractions.

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Fry CH, Young JS, Jabr RI, McCarthy C, Ikeda Y, Kanai AJ. Modulation of spontaneous activity in the overactive bladder: the role of P2Y agonists. Am J Physiol Renal Physiol 302: F1447-F1454, 2012. First published February 22, 2012 doi:10.1152/ajprenal.00436.2011Spinal cord transection (SCT) leads to an increase in spontaneous contractile activity in the isolated bladder that is reminiscent of an overactive bladder syndrome in patients with similar damage to the central nervous system. An increase in interstitial cell number in the suburothelial space between the urothelium and detrusor smooth muscle layer occurs in SCT bladders, and these cells elicit excitatory responses to purines and pyrimidines such as ATP, ADP, and UTP. We have investigated the hypothesis that these agents underlie the increase in spontaneous activity. Rats underwent lower thoracic spinal cord transection, and their bladder sheets or strips, with intact mucosa except where specified, were used for experiments. Isometric tension was recorded and propagating Ca 2ϩ and membrane potential (Em) waves were recorded by fluorescence imaging using photodiode arrays. SCT bladders were associated with regular spontaneous contractions (2.9 Ϯ 0.4/min); ADP, UTP, and UDP augmented the amplitude but not their frequency. With strips from such bladders, a P2Y 6-selective agonist (PSB0474) exerted similar effects. Fluorescence imaging of bladder sheets showed that ADP or UTP increased the conduction velocity of Ca 2ϩ /Em waves that were confined to regions of the bladder wall with an intact mucosa. When transverse bladder sections were used, Ca 2ϩ /Em waves originated in the suburothelial space and propagated to the detrusor and urothelium. Analysis of wave propagation showed that the suburothelial space exhibited properties of an electrical syncitium. These experiments are consistent with the hypothesis that P2Y-receptor agonists increase spontaneous contractile activity by augmenting functional activity of the cellular syncitium in the suburothelial space.spinal cord injury; spontaneous contraction; Ca 2ϩ waves; ADP THE OVERACTIVE BLADDER IN patients is associated with spontaneous transient increases in pressure that occur particularly during filling and, if sufficiently severe, contribute to lower urinary tracts symptoms of frequency, urgency, and incontinence (1). The origins of this common pathology are unclear but can be mimicked in animals in which the bladder outflow is artificially obstructed or the spinal cord is transected (SCT; T8 -T9) (12). Whole rat bladders from an SCT model demonstrate large, regular contractions (ϳ2/min) that coincide with waves of raised intracellular Ca 2ϩ and depolarization (E m ) propagating across the surface but originating from one or very few foci. This is in contrast to normal animals where more frequent (5-10/min), low-amplitude contractions are associated with multiple Ca 2ϩ /E m foci (12,14). The detrusor layer of the bladder wall contributes the bulk of tissue and hence most of the Ca 2ϩ /E m signals, but an in...