Spontaneous activity of lower urinary tract smooth muscles: correlation between ion channels and tissue function

Smooth muscles from the urethra and bladder display characteristic patterns of spontaneous contractile activity in the filling phase of the micturition cycle. Tonic contractions are seen in the urethral smooth muscles, and phasic contractions occur in the detrusor. Overactivity in the detrusor is a common clinical problem. The ion channels in the smooth muscle membranes play an important role in determining the functional properties, and are obvious targets for treatment of the overactive bladder. Recent evidence suggests that interstitial cells may also play a role in determining the pattern of spontaneous activity, although their precise role is less well established in the urinary tract than in the gut. The ion channels involved in these cells are also of interest. This review discusses what is known of ion channels in these tissues, and their implications for function.     

Carbachol-Induced Volume Adaptation in Mouse Bladder and Length Adaptation via Rhythmic Contraction in Rabbit Detrusor

The length-tension (L-T) relationships in rabbit detrusor smooth muscle (DSM) are similar to those in vascular and airway smooth muscles and exhibit short-term length adaptation characterized by L-T curves that shift along the length axis as a function of activation and strain history. In contrast to skeletal muscle, the length-active tension (L-T (a)) curve for rabbit DSM strips does not have a unique peak tension value with a single ascending and descending limb. Instead, DSM can exhibit multiple ascending and descending limbs, and repeated KCl-induced contractions at a particular muscle length on an ascending or descending limb display increasingly greater tension. In the present study, mouse bladder strips with and without urothelium exhibited KCl-induced and carbachol-induced length adaptation, and the pressure-volume relationship in mouse whole bladder displayed short-term volume adaptation. Finally, prostaglandin-E(2)-induced low-level rhythmic contraction produced length adaptation in rabbit DSM strips. A likely role of length adaptation during bladder filling is to prepare DSM cells to contract efficiently over a broad range of volumes. Mammalian bladders exhibit spontaneous rhythmic contraction (SRC) during the filling phase and SRC is elevated in humans with overactive bladder (OAB). The present data identify a potential physiological role for SRC in bladder adaptation and motivate the investigation of a potential link between short-term volume adaptation and OAB with impaired contractility.     

Urinary bladder instability induced by selective suppression of the murine small conductance calcium-activated potassium (SK3) channel

Small conductance, calcium-activated potassium (SK) channels have an important role in determining the excitability and contractility of urinary bladder smooth muscle. Here, the role of the SK isoform SK3 was examined by altering expression levels of the SK3 gene using a mouse model that conditionally overexpresses SK3 channels (SK3^T/T). Prominent SK3 immunostaining was found in both the smooth muscle (detrusor) and urothelium layers of the urinary bladder. SK currents were elevated 2.4-fold in isolated myocytes from SK3^T/T mice. Selective suppression of SK3 expression by dietary doxycycline (DOX) decreased SK current density in isolated myocytes, increased phasic contractions of isolated urinary bladder smooth muscle strips and exposed high affinity effects of the blocker apamin of the SK isoforms (SK1-3), suggesting an additional participation from SK2 channels. The role of SK3 channels in urinary bladder function was assessed using cystometry in conscious, freely moving mice. The urinary bladders of SK3^T/T had significantly greater bladder capacity, and urine output exceeded the infused saline volume. Suppression of SK3 channel expression did not alter filling pressure, threshold pressure or bladder capacity, but micturition pressure was elevated compared to control mice. However, SK3 suppression did eliminate excess urine production and caused a marked increase in non-voiding contractions. The ability to examine bladder function in mice in which SK3 channel expression is selectively altered reveals that these channels have a significant role in the control of non-voiding contractions in vivo . Activation of these channels may be a therapeutic approach for management of non-voiding contractions, a condition which characterizes many types of urinary bladder dysfunctions including urinary incontinence.     

Interaction between interstitial cells and smooth muscles in the lower urinary tract and penis

Smooth muscles in the lower urinary tract and corporal tissue exhibit spontaneous contractile activity which depends on L-type Ca²⁺ channels. The mechanism underlying this activity is spontaneous electrical activity which shows varied form and property between these tissues. Recent studies revealed that interstitial cells (ICs) are widely distributed in the genitourinary system, and suggested their involvement in spontaneous muscle activity. ICs in the system are not a simple analogy of interstitial cells of Cajal (ICC) in the gut, which act as electrical pacemaker, but represent variability amongst tissues which may account for individual characteristics of each organ. In the bladder and corporal tissue, where smooth muscle cells are capable of generating spontaneous electrical activity, ICs may modulate smooth muscle activity. ICs in corporal tissue release prostaglandins via cyclooxygenase-2 (COX-2) activity and reinforce not only spontaneous but also nerve-mediated α-adrenergic contractions. In the bladder, their fundamental role in the integration of signals between populations of cells has been proposed, and thus changes in ICs may contribute to an overactive bladder, a pathological condition which results from increased excitability in detrusor smooth muscles. In the urethra, ICs may act as electrical pacemakers as do ICC. However, overall contractility of urethral smooth muscles does not necessarily rely on pacemaking of ICs, and thus some population of smooth muscles may also have their own excitability.     

Physiological and pathophysiological implications of micromotion activity in urinary bladder function

‘Micromotions’ is a term signifying the presence of localized microcontractions and microelongations, alongside non‐motile areas. The motile areas tend to shift over the bladder surface with time, and the intravesical pressure reflects moment‐by‐moment summation of the interplay between net contractile force generated by micromotions and general bladder tone. Functionally, the bladder structure may comprise modules with variable linkage, which supports presence of localized micromotions (no functional linkage between modules), propagating contractions (where emergence of linkage allows sequential activation) and the shifting of micromotions over time. Detrusor muscle, interstitial cells and intramural innervation have properties potentially relevant for initiating, coordinating and modulating micromotions. Conceptually, such activity could facilitate the generation of afferent activity (filling state reporting) in the absence of intravesical pressure change and the ability to transition to voiding at any bladder volume. This autonomous activity is an intrinsic property, seen in various experimental contexts including the clinical setting of human (female) overactive bladder. ‘Disinhibited autonomy’ may explain the obvious micromotions in isolated bladders and perhaps contribute clinically in neurological disease causing detrusor overactivity. Furthermore, any process that could increase the initiation or propagation of microcontractions might be anticipated to have a functional effect, increasing the likelihood of urinary urgency and detrusor overactivity respectively. Thus, models of bladder outlet obstruction, neurological trauma and ageing provide a useful framework for detecting cellular changes in smooth muscle, interstitial cells and innervation, and the consequent effects on micromotions.     

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.     

Mechanotransduction of extracellular signal-regulated kinases 1 and 2 mitogen-activated protein kinase activity in smooth muscle is dependent on the extracellular matrix and regulated by matrix metalloproteinases

Excessive wall stretch of distensible hollow organs in cardiovascular and urinary systems can activate matrix metalloproteinases (MMPs), thereby releasing matrix neoepitopes and growth factor ligands, leading to ERK1/2 activation. However, the role of MMPs in mechanotransduction of ERK1/2 signaling in the bladder is unknown. We examined bladders undergoing sustained distension over time, which provides a novel platform for smooth muscle mechanotransduction studies. Bladder distension ex vivo caused increased proliferation and MMP activity. Conditioned medium from distended compared with undistended bladders induced proliferation in bladder smooth muscle cells (BSMCs). When conditioned medium from distended bladders was used to proteolyze collagen type I matrices, matrices augmented BSMC proliferation, which was inhibited if bladders were distended in presence of broad-spectrum MMP inhibitors. Distension of ex vivo bladders also induced ERK1/2 phosphorylation in situ, which was dependent on MMP activity in the intact bladder. Similarly, stretching BSMCs in vitro induced increases in ERK1/2 activation and ERK1/2-dependent proliferation under discrete mechanical conditions, and distension conditioned medium itself induced MMP-dependent ERK1/2 activation in BSMCs. Overall, stretch-induced proliferation and ERK1/2 signaling in bladder tissue and BSMCs likely depend on secreted MMP activity. Identification of intermediaries between MMPs and ERK1/2 may elaborate novel mechanisms underlying mechanotransduction in bladder smooth muscle.     

Transplantation of preserved human amniotic membrane for bladder augmentation in rats

Although gastrointestinal segments have been widely used for bladder reconstruction, they are not ideal because of the possible complications. Searches have therefore continued for an alternative material for augmentation. Here, we performed bladder augmentation in rats using human amniotic membrane (hAM). Morphologically, the hAM-augmented bladder revealed regeneration of urothelium, detrusor smooth muscle, and nerve fibers within 3 months post-operatively. In our functional evaluation of bladder strips, we compared hAM-augmented bladders with bladders augmented using small intestinal submucosa (SIS). For example, at 6 months post-operatively, contractions of the following size (as a percentage of the responses in the control-bladder group) were obtained in response to high potassium, carbachol, and electrical field stimulation, respectively: hAM 22% vs SIS 15%, hAM 15% vs SIS 7%, hAM 5.3% vs SIS 1.3% (no significant differences, hAM vs SIS). Both hAM- and SIS-augmented bladders displayed adequate capacity and compliance. The present results indicate that, for bladder augmentation, hAM can be used as a scaffold and is comparable in this respect with SIS. hAM can be more easily obtained than SIS and requires little preparation, and its use raises few ethical questions. Hence, hAM may represent a new therapeutic alternative for urological reconstructions.     

Urinary bladder smooth muscle engineered from adipose stem cells and a three dimensional synthetic composite

Human adipose stem cells were cultured in smooth muscle inductive media and seeded into synthetic bladder composites to tissue engineer bladder smooth muscle. 85:15 Poly-lactic-glycolic acid bladder dome composites were cast using an electropulled microfiber luminal surface combined with an outer porous sponge. Cell-seeded bladders expressed smooth muscle actin, myosin heavy chain, calponinin, and caldesmon via RT-PCR and immunoflourescence. Nude rats (n = 45) underwent removal of half their bladder and repair using: (i) augmentation with the adipose stem cell-seeded composites, (ii) augmentation with a matched acellular composite, or (iii) suture closure. Animals were followed for 12 weeks post-implantation and bladders were explanted serially. Results showed that bladder capacity and compliance were maintained in the cell-seeded group throughout the 12 weeks, but deteriorated in the acellular scaffold group sequentially with time. Control animals repaired with sutures regained their baseline bladder capacities by week 12, demonstrating a long-term limitation of this model. Histological analysis of explanted materials demonstrated viable adipose stem cells and increasing smooth muscle mass in the cell-seeded scaffolds with time. Tissue bath stimulation demonstrated smooth muscle contraction of the seeded implants but not the acellular implants after 12 weeks in vivo. Our study demonstrates the feasibility and short term physical properties of bladder tissue engineered from adipose stem cells.     

Altered urinary bladder function in mice lacking the vanilloid receptor TRPV1

In the urinary bladder, the capsaicin-gated ion channel TRPV1 is expressed both within afferent nerve terminals and within the epithelial cells that line the bladder lumen. To determine the significance of this expression pattern, we analyzed bladder function in mice lacking TRPV1. Compared with wild-type littermates, trpv1(-/-) mice had a higher frequency of low-amplitude, non-voiding bladder contractions. This alteration was accompanied by reductions in both spinal cord signaling and reflex voiding during bladder filling (under anesthesia). In vitro, stretch-evoked ATP release and membrane capacitance changes were diminished in bladders excised from trpv1(-/-) mice, as was hypoosmolality-evoked ATP release from cultured trpv1(-/-) urothelial cells. These findings indicate that TRPV1 participates in normal bladder function and is essential for normal mechanically evoked purinergic signaling by the urothelium.     

What is the hair cell transduction channel?

Rhythmic electrical activity is a feature of most smooth muscles but the mechanical consequences can vary from regular rapid phasic contractions to sustained contracture. For many years it was thought that spontaneous electrical activity originated in smooth muscle cells but recently it has become apparent that there are specialized pacemaker cells in many organs that are morphologically and functionally distinct from smooth muscle and that the former cells are the source of spontaneous electrical activity. Such a pacemaker function is well documented for the ICC of the gastrointestinal tract but evidence is accumulating that ICC-like cells play a similar role in other types of smooth muscle. We have recently shown that there are specialized pacemaking cells in the rabbit urethra which are spontaneously active when freshly isolated, readily distinguishable from smooth muscle cells under bright field illumination and relatively easy to study using patch-clamp and confocal imaging techniques. Recent results suggest that calcium oscillations in isolated rabbit urethral interstitial cells are initiated by calcium release from ryanodine sensitive intracellular stores, that oscillation frequency is very sensitive to the external calcium concentration and that conversion of the primary oscillation to a propagated calcium wave depends upon IP₃-induced calcium release.     

Smooth muscle hypertrophy following partial bladder outlet obstruction is associated with overexpression of non-muscle caldesmon

Partial bladder outlet obstruction (PBOO) induces remodeling of urinary bladder smooth muscle (detrusor). We demonstrate an increase in bladder wall mass, muscle bundle size, and a threefold increase in the cross-sectional area of detrusor myocytes following PBOO in male New Zealand White rabbits compared to that of controls. Some bladders with detrusor hypertrophy function close to normal (compensated), whereas others were dysfunctional (decompensated), showing high intravesical pressure, large residual urine volume, and voiding difficulty. We analyzed the expression of smooth muscle-specific caldesmon (h-CaD) and non-muscle (l-CaD) by Western blotting, RT-PCR, and real-time PCR. The expression of l-CaD is increased significantly at the mRNA and protein levels in the decompensated bladders compared to that of normal and compensated bladders. The CaD was also co-localized with myosin containing cytoplasmic fibrils in cells dissociated from obstructed bladders and cultured overnight. Our data show that the inability of decompensated bladders to empty, despite detrusor hypertrophy, is associated with an overexpression of l-CaD. The level of l-CaD overexpression might be a useful marker to estimate the degree of detrusor remodeling and contractile dysfunction in PBOO.     

Autonomic nervous influence of the female guinea-pig urinary bladder during the oestrus cycle

The mammalian urinary bladder receives dual innervation. The excitatory innervation is considered to be partly cholinergic and partly mediated via NANC-receptors. Several (co-)transmitters have been suggested. The adrenergic inhibitory innervation is mediated via α- and β-receptors. Female sex hormones could change autonomic influence of urogenital organs. It was considered to be of interest to characterize the spontaneous and nerve stimulation-induced muscular activity in the urinary bladder of the female guinea-pig during the oestrus cycle. Both the spontaneous activity and nerve-induced activity varied according to the hormonal status of the animal. An α-adrenergic inhibitory influence was identified. It was further confirmed that the excitatory innervation could not be blocked by the cholinergic antagonist scopolamine, while α-β-methylene ATP partly inhibited nerve stimulation-induced smooth muscle response, most prominent at cycle day 6. Indomethacin did not impair spontaneous activity or nerve stimulation-induced activity. Nitric oxide reduced nerve stimulation-induced responses on cycle day 12. Imperative urinary bladder contractions are reported to diminish after oestrogen use and in the female a hormonal effect of the nervous influence on the urinary bladder smooth muscle is suggested.     

Evidence for the involvement of the cyclooxygenase-metabolic pathway in diclofenac-induced inhibition of spontaneous contraction of rat portal vein smooth muscle cells.

The effects of diclofenac, a cyclooxygenase (COX) inhibitor, were investigated on spontaneous phasic contractions of longitudinal preparations of the rat portal vein. Diclofenac produced a concentration-dependent decrease in the amplitude of these spontaneous phasic contractions. Diclofenac (30 microM) decreased the amplitude of the spontaneous phasic increase in the F340/F380 ratio of Fura PE3, an indicator of intracellular Ca2+ concentration. It also reduced the number of action potentials in each burst discharge without changing the resting membrane potential of longitudinal smooth muscle cells. The extent of the distribution of Lucifer Yellow injected into a smooth muscle cell was decreased in the presence of diclofenac (30 microM). Both AH6809, a prostanoid EP receptor antagonist, and SQ22536, an adenylate cyclase inhibitor, decreased the amplitude of the spontaneous contractions. On the other hand, neither ozagrel, a thromboxane synthase inhibitor, nor SQ29548, a prostanoid TP receptor antagonist, significantly affected spontaneous contractions. These results indicate that diclofenac inhibits the amplitude of spontaneous contractions of the rat portal vein through inhibition of electrical activity, which may be related to an inhibition of the cyclooxygenase pathway.     

Proteases of the elastase type

Elastin in the most resistant fibrous protein of the organisms. Its degradation is catalysed by proteases designated as elastases. Elastic fibers appeared during phylogenesis at the level of the first Vertebrates and rendered possible the emergence of efficient circulatory and respiratory systems which were necessary for the development of the higher Vertebrates. Several pathological conditions, mostly age-dependent, are accompanied by the degradation of elastic fibers or their alteration due to increasing association with lipids and calcium salts. Several proteases (endopeptidases) of cellular origin were described over the last years, especially those of PMN leukocytes, platelets, monocytes-macrophages, smooth muscle cells and fibroblasts. Although less active on fibrous elastin than pancreatic elastase, these enzymes may well play an important role in the development of age-dependent pathologies such as athero-arteriosclerosis and emphysema. The involvement of cellular elastases in these pathologies is discussed in some detail. The age-dependent increase, both in vivo and in vitro of the elastase activity of fibroblasts and smooth muscle cells appears to play an important role in the modifications of cell behaviour observed in the above pathologies.     

Neurotensin in the human fallopian tube: Immunohistochemical localization and effects of synthetic neurotensin on motor activity in vitro

With the use of different region-specific antisera and the peroxidase-antiperoxidase (PAP) technique, neurotensin-immunoreactivity was found in nerve fibers of the human fallopian tube. Neurotensin-immunoreactive fibers occurred at blood vessels and in contact with smooth muscle cells of the muscular stratum. In vitro experiments with helical strips of the myometrium revealed dose-dependent excitatory actions of neurotensin on resting tension as well as on amplitude and frequency of spontaneous contractions. The results may suggest a neurotransmitter function of neurotensin in the regulation of human oviductal smooth muscle activity. Thus, neurotensin may be essential in the transport of the eggs through the fallopian tube.     

Morphological analysis of the acute effects of overdistension on the extracellular matrix of the rat urinary bladder wall.

PURPOSE: To investigate the morphological effects of acute overdistension in the structure of the extracellular matrix of the bladder wall in rats. MATERIALS AND METHODS: The bladders of a group of 6 male Wistar rats were transurethrally overdistended for 3 hours. Another identical group (the control group) was only submitted to a sham operation. Specimens from the bladder dome were analyzed with light microscopy (LM), transmission electron microscopy (TEM) and scanning electron microscopy (SEM). RESULTS: LM--The control group bladders had a 4 to 5 layer urothelium, a lamina propria, and a smooth muscle layer with longitudinal and transversal fibers. The overdistended bladders presented an intense interstitial infiltrate in the lamina propria, and a less intense infiltrate among the smooth muscle fibers. TEM--The cells of the overdistended bladders had a significant amount of vacuoles, unlike the control bladders, where such vacuoles were scarce or absent. SEM--A delicate three-dimensional mesh of collagen fibrils was observed in the lamina propria of the bladder walls from the control group. Whilst for the control group this mesh consisted of distinct geometric structures, with mostly circular cellular spaces surrounded by the fibrils, the overdistended group showed evidence of distortion of the mesh, with flattened and elongated cellular spaces. CONCLUSIONS: Acute bladder overdistension induces structural modifications, altering the arrangement and interaction of collagen fibrils, as well as incipient tissue damage as edema in the lamina propria and smooth muscle layers.     

Pyeloureteral motility and ureteral peristalsis: essential role of sensory nerves and endogenous prostaglandins

The cellular mechanisms that underlie the initiation and propagation of the peristaltic contractions, which transport urine from the kidney to the bladder for storage, remain little understood. Extracellular and intracellular microelectrode recordings have identified two populations of smooth muscle cells as well as a population of renal interstitial cells (RICs) that all display spontaneous electrical activity. By analogy with the heart it has been proposed that atypical smooth muscle cells, preferentially located in the very proximal regions of the renal pelvis, generate the essential pacemaker signal. These pacemaker potentials propagate to neighbouring typical smooth muscle cells or RICs to trigger action potential discharge. These action potentials then propagate distally to trigger other bundles of typical smooth muscle cells. The frequency of action potential discharge and contraction decreases as the relative number of RICs and atypical smooth muscle cells compared to typical smooth muscle cells decreases with distance from the renal fornix. It is clear that functional capsaicin-sensitive sensory afferents and the endogenous release of both tachykinins and prostaglandins are essential in the maintenance of normal peristalsis, as well as in monitoring and responding to any chemical or mechanical stimulation. However, the cellular mechanisms underlying the action of these endogenously-released agents remain to be elucidated.     

Effect of ageing on the response of the rat isolated testicular capsule to norepinephrine, acetylcholine and prostaglandins

The response of the rat isolated testicular capsule to norepinephrine (NE), acetylcholine (ACh) and prostaglandins (PG) was found to be age-dependent. The testicular capsular contractions attributable to smooth muscle cells located within the tunica albuginea may be involved in the transport of non-motile spermatozoa from the testis and into the epididymis. With an increase in age of the age of the rat there was a concomitant increase in mass of the testis, thickness of the capsule and the amount of smooth muscle fibers present in the tunica albuginea. The response to the addition of equimolar concentrations of NE and PGA2 became progressively greater as the age of the animal increased. However, the response of the testicular capsule to ACh remained constant throughout adulthood, while PGF2 alpha became progressively less stimulatory as the age of the animal increased. These findings suggest that neurohumoral agents may have an important role in the maintenance of testicular capsular contractions with regard to ageing.     

Autoantibody-mediated bladder dysfunction in type 1 diabetes

Bladder dysfunction is a common complication of diabetic autonomic neuropathy; however, its cause remains uncertain. We have recently identified a novel IgG autoantibody (Ab) in patients with type 1 diabetes that acts as an agonist at the dihydropyridine (DHP) site of L-type voltage-gated calcium channels (VGCC), disrupting neuronal regulation of visceral smooth muscle. In the present study, passive transfer to mice of IgG from patients with type 1 diabetes was used to investigate the role of anti-VGCC Abs in mediating diabetic bladder dysfunction. Injection of mice with diabetic immunoglobulin (IgG) with anti-VGCC activity induced features of an overactive bladder, including phasic detrusor contractions and a loss of bladder wall compliance. The bladder overactivity is mimicked by the DHP agonist Bay K8644, reversed by the DHP antagonist nicardipine, but is insensitive to the motor nerve blocker tetrodotoxin, indicating that the anti-VGCC Ab acts at the level of the bladder detrusor itself. This study reports the first evidence of Ab-mediated bladder dysfunction in type 1 diabetes, which may be part of a wider spectrum of smooth muscle and cardiac abnormalities.