Neuronal-derived nitric oxide modulates the activity of mouse detrusor smooth muscle.

AIMS: We investigated the roles of neuronal-derived nitric oxide (NO) in the modulation of spontaneous activity of mouse detrusor smooth muscle. METHODS: Detrusor smooth muscle strips were isolated from nNOS gene knock-out (nNOS(-/-) ) mice and their wild type siblings (nNOS(+/+) ). The properties of smooth muscle cells were assessed using intracellular electrophysiology and Ca(2+) imaging by laser-scanning confocal microscopy. The effects of an nNOS inhibitor, 7-nitro indazole (7-NI) on electrically evoked contractility were assessed using nNOS(+/+) mouse detrusor strips. RESULTS: In spontaneously active cells, the frequency of spontaneous action potentials (sAPs) and whole cell Ca(2+) flashes in nNOS(-/-) preparations was lower than that in the nNOS(+/+) preparations. The frequency of sAPs was enhanced by a nitric oxide donor, diethylamine NONOate sodium salt (NONOate; 100 µM), both when used alone and when the cGMP pathway was blocked by 1H-[1,2,4] oxadiazolo [4,3-a] quinoxalin-1-one (ODQ, 10 µM). 7-NI (100 µM) significantly suppressed the electrically evoked contraction of mouse detrusor strips. CONCLUSIONS: We suggest that neuronal-derived NO facilitates the generation of spontaneous activity via a cGMP-independent pathway, and consequently enhances the evoked contraction of detrusor. Dysregulation of nNOS containing nerves may underlie bladder pathologies.

Ion channel modulators and urinary tract function.

The membrane potential fulfils an important role in initiating smooth muscle contraction, through its depolarization and the subsequent influx of Ca(2+) through voltage-gated Ca(2+) channels. Changes in membrane potential can also coordinate contraction across great distances, utilizing the speed of electrical current flow through gap junctions. Hence, regulating membrane potential can greatly influence smooth muscle function. In this chapter, we will consider the influence of ion channels, as dynamic gatekeepers of membrane permeability, on urogenital function. Through their ability to act as key regulators of both the resting membrane potential and its dynamic changes, they provide important pharmacological targets for influencing urogenital function.Urogenital smooth muscle and urothelia contain a diverse range of molecularly and functionally distinct K(+) channels, which are key to regulating the resting membrane and for re-establishing the normal membrane potential following both active and passive changes. The voltage-gated Ca(2+) channels are key to initiating contraction and causing rapid depolarization, supplemented in some smooth muscles by rapid Na(+) conductances. The Cl(-) channels, often assumed to be passive, can actively change the membrane potential, and hence, cellular function, because Cl(-) is not usually at its equilibrium potential. The useful ways in which these ion channels can be targeted therapeutically in the ureter, bladder and urethra are discussed, focussing particularly on treatments for ureteric obstruction and detrusor overactivity. Current treatments for many urinary tract disorders, particularly the overactive bladder, are complicated by side effects. While ion channels have traditionally been considered as poor therapeutic targets by the pharmaceutical industry, our increasing knowledge of the molecular diversity of K(+) and Cl(-) channels gives new hope for more narrowly focused drug targeting, while the exciting discoveries of active currents in interstitial cells give us a new set of cellular targets for drugs.

Functional coordination of motor activity in colonic and recto-anal smooth muscles in rat experimental model.

Spontaneous or electrically elicited contraction and/or relaxation of the longitudinal and circular muscles of the colon and rectum and the anal canal in rat segment preparations were recorded simultaneously to display contractile potency and functional coordination of muscles in the large intestine. Spontaneous high-amplitude contractions, but not relaxations, appeared synchronously in the longitudinal and circular muscles of the colon and rectum. The anal canal showed contractions following the activity of rectal muscles. The colonic and rectal longitudinal muscle responded to electrical stimulation with frequency-dependent contraction. The response of the circular muscle of the colon initially consisted of frequency-independent relaxation followed by frequency-dependent contraction, which was more pronounced in the distal colon, while the rectal circular muscle responded with contraction. The responses appeared synchronously, demonstrating the coactivation of the nervous pathways that supply both muscles. The contractions of longitudinal muscles were more pronounced suggesting a dominant role of this layer in the coordinated motor activity. The local response of the internal anal sphincter was biphasic, comprising short contractions followed by relaxation, while the response of the anal canal was contraction. The contractile local responses increased from the colon to the rectum, while differences in the relaxations were not observed, indicating rather higher contractile potency than the relaxation ability of muscles in the distal part of the gut.

A survey of commonalities relevant to function and dysfunction in pelvic and sexual organs.

Micturition, defecation and sexual function are all programmed through spinal reflexes that are under descending control from higher centres. Interaction between these reflexes can clearly be perceived, and evidence is accumulating the dysfunction in one reflex is often associated with dysfunction in another. In this article, we describe some of the basic properties and neural control of the smooth muscles mediating the reflexes, reviewing the common features that underlie these reflex functions, and what changes may be responsible for dysfunction. We propose that autonomic control within the pelvis predisposes pelvic and sexual organs to crosstalk, with the consequence that diseases and conditions of the pelvis are subject to convergence on a functional level. It should be expected that disturbance of the function of one system will inevitably impact adjacent systems.

Evolving mechanisms of action of alverine citrate on phasic smooth muscles.

BACKGROUND AND PURPOSE: We have investigated the mechanisms underlying the paradoxical ability of the antispasmodic, alverine, to enhance spontaneous activity in smooth muscles while suppressing evoked activity. EXPERIMENTAL APPROACH: The effects of alverine on spontaneous and induced contractile activity were examined in preliminary experiments with various smooth muscles. More detailed effects were also investigated by recording membrane potential, intracellular Ca2+ concentration ([Ca2+]i) and tension from single-bundle detrusor smooth muscle (DSM) of the guinea-pig urinary bladder. KEY RESULTS: Alverine (10 microM) increased the frequency and amplitude of spontaneous action potentials, transient increases in [Ca2+]i and associated contractions. Alverine also decreased action potential rate of decay, suggesting inhibition of L-type Ca channel inactivation. Charybdotoxin (50 nM) but neither cyclopiazonic acid (10 microM) nor Bay K 8644 (10 microM) attenuated alverine-induced enhancement of spontaneous contractions. Alverine suppressed contractions produced by high K (40 mM) or ACh (10 microM), without affecting electrical responses and with little suppression of increases in [Ca2+]i. This feature was very similar to that of the effects of the Rho kinase inhibitor Y-27632 (10 microM). CONCLUSIONS AND IMPLICATIONS: Alverine may increase Ca influx during action potentials due to inhibition of the inactivation of L-type Ca channels, but may also suppress evoked activity by inhibiting the sensitivity of contractile proteins to Ca2+. The proportional contribution of Ca-dependent and Ca-independent contractions in DSM may differ between spontaneous and evoked activity, necessitating further investigations into the interactions between these pathways for assessing the therapeutic potential of alverine to treat DSM dysfunction.

Mechanisms controlling normal defecation and the potential effects of spinal cord injury.

Spinal cord injury frequently leads to bowel dysfunction with the result that emptying the bowel can occupy a significant part of the day and reduce the quality of life. This chapter contains an overview of the function and morphology of the normal distal gut in the human, and of gut behaviour in normal defecation. In humans, this can be monitored and is described, but knowledge of the mechanisms controlling it is limited. Work on animals has shown that the intrinsic activity of the smooth muscles and their interactions with the enteric nervous system can program the activity that is necessary to expel waste material, but the external anal sphincter is controlled through somatic nerves. The gut however also receives input from the central nervous system through autonomic nerves, and a spinal reflex centre exists. Voluntary effort to induce defecation can influence all the control mechanisms, but the precise importance of each is not understood. The behaviour and properties of the individual muscles in the normal human rectum and anal canal are described, including their responses to intrinsic nerve stimulation and adrenergic and cholinergic agonists. The effects of established spinal cord injury are then considered. For convenience, supraconal and conal/cauda equina lesions are considered as two categories. Prolongation of transit times and disordered defecation are common problems. Supraconal lesions result in reduced resting anal pressures and increased risk of fecal incontinence. The acute effects of spinal cord injury are described, with injury causing ileus (prolonged total gastrointestinal transit times), constipation (prolonged colonic transit times) and fecal incontinence (passive leakage).

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.

Mechanism of action of botulinum toxin on the internal anal sphincter.

BACKGROUND: Botulinum toxin is an effective treatment for anal fissure. Manometric studies support an apparent action of botulinum toxin on the internal anal sphincter (IAS). This aim of this study was to establish the underlying mechanism. METHODS: Porcine IAS strips were suspended in a superfusion organ bath and allowed to equilibrate. Electrical field stimulation (EFS) was applied with parameters that induced nitrergic relaxation followed by noradrenaline-mediated contraction. These responses were compared before and after addition of botulinum toxin. RESULTS: All strips developed myogenic tone, which was slightly increased following the addition of botulinum toxin. EFS-induced nitrergic relaxation was unaffected by toxin treatment. However, EFS-induced contraction was significantly reduced by toxin treatment. 1,1-dimethyl-4-phenylpiperazinium iodide (DMPP), a nicotinic agonist, caused muscle strip contraction, which was blocked by guanethidine, implying the presence of sympathetic ganglia within the IAS. Botulinum toxin significantly attenuated DMPP-induced contraction. CONCLUSION: In the treatment of anal fissure the major effect of botulinum toxin on the IAS is blockade of sympathetic (noradrenaline mediated) neural output. This is probably a postganglionic action, involving a reduction in noradrenaline release at the neuromuscular junction. Botulinum toxin has no significant effect on nitrergic transmission, which is probably not vesicular in nature.

Multiple effects of mefenamic acid on K(+) currents in smooth muscle cells from pig proximal urethra.

The effects of mefenamic acid on both membrane potential and K+ currents in pig urethral myocytes were investigated using patch-clamp techniques (conventional whole-cell, cell-attached, outside-out and inside-out configuration). In the current-clamp mode, mefenamic acid caused a concentration-dependent hyperpolarization, which was inhibited by preapplication of 1 microm glibenclamide. In the voltage-clamp mode, mefenamic acid induced an outward current that was blocked by glibenclamide even in the presence of iberiotoxin (IbTX, 300 nm) at -50 mV. ATP-sensitive K+ channels (KATP channels) could be activated in the same patch by mefenamic acid and levcromakalim, with the same unitary amplitude and the similar opening gating at -50 mV in cell-attached configuration. In outside-out recording, external application of mefenamic acid activated intracellular Ca2+-activated IbTX-sensitive large-conductance K+ channels (BKCa channels). Mefenamic acid (or=100 microm) increased sustained outward currents, diminishing the activity of STOCs. Over the whole voltage range, mefenamic acid caused opposite effects on the membrane currents in the absence and presence of 5 microm glibenclamide. In the presence of 10 mm 4-aminopyridine (4-AP), mefenamic acid only increased the outward currents. These results indicate that mefenamic acid increases the channel activities of two distinct types of K+ channels (i.e. BKCa channels and KATP channels) and decreased 4-AP-sensitive K+ channels in pig urethral myocytes.

Botulinum toxin injection inhibits myogenic tone and sympathetic nerve function in the porcine internal anal sphincter.

OBJECTIVE: Botulinum toxin is an effective treatment for anal fissure, though there is a lack of agreement over the optimal site for its injection. This reflects our current ignorance of its mechanism, and whether it has any action on the nerves of the internal anal sphincter (IAS). This study set out to resolve this issue through use of a pig model. MATERIALS AND METHODS: Eight pigs were studied in pairs: one of each pair received a botulinum toxin injection into the anal sphincter, whilst the other acted as its control. Manometry was performed every two weeks under anaesthesia. Pigs were slaughtered at between four and six weeks after injection and the properties of the IAS compared in vitro. RESULTS: Whilst maximum anal resting pressure (MARP) increased slowly in control pigs during the experimental period, reflecting weight gain, a fall was observed in treated pigs. In vitro, IAS strips from control pigs generated 400 mg of spontaneous tone per gram of tissue (+/- 45; standard error), compared to 250 (+/- 25) mg/g tissue from treated pigs (P < 0.01). Electric Field Stimulation at 50 Hz produced 150 (+/- 22) mg contraction/gram tissue in IAS strips from control pigs compared to 53 (+/- 13) mg/g tissue in treated pigs (P < 0.0005). This contractile response was blocked by guanethidine. CONCLUSION: Botulinum toxin has a significant action on the IAS. It reduces myogenic tone and contractile responses of this tissue to sympathetic nerve stimulation. Further studies are required to clarify its mechanism of action more precisely.

L-Erythro-methoxamine is more potent than phenylephrine in effecting contraction of internal anal sphincter in vitro.

BACKGROUND: Topical phenylephrine has been shown to increase resting anal canal pressure in normal and incontinent individuals. However, high concentrations of gel (10-40 per cent) are required that may cause local side-effects. The aim of this study was to determine whether methoxamine, another alpha-1-adrenoceptor agonist, might be a more potent alternative to phenylephrine. METHODS: Porcine internal anal sphincter (IAS) tissue was cut into strips, suspended in a superfusion organ bath and allowed to equilibrate. Strips were subjected to each drug under test for 20 s, sufficient to obtain stable tone. Phenylephrine, methoxamine (1 : 1 : 1 : 1 ratio of its four isomers) and each of the individual isomers of methoxamine were evaluated in turn. RESULTS: In vitro, methoxamine racemate and phenylephrine were similarly potent in causing contraction of IAS strips (mean(s.e.m.) dose giving half maximal effect (EC(50)) at 74.7(16.5) versus 58.3(13.4) micro M respectively; P = 0.443). However, one of the methoxamine isomers, L-erythro-methoxamine (EC(50) 17.6(3.7) micro M), was significantly more potent than the other three isomers, methoxamine racemate and phenylephrine (P = 0.002). CONCLUSION: L-Erythro-methoxamine is four times more potent than phenylephrine and is a possible treatment for incontinence. Trials are under way to examine the efficacy of L-erythro-methoxamine in vivo.

Innervation of the detrusor muscle bundle in neurogenic detrusor overactivity.

OBJECTIVE: To evaluate the peripheral anatomical distribution of innervation within muscle bundles of the detrusor and the changes arising in neurogenic detrusor overactivity (DO). PATIENTS AND METHODS: Full-thickness samples from the bladder dome of three cadaveric transplant organ donors and four people with neurogenic DO caused by spinal cord injury were compared. Systematic serial cryostat sections were stained using Masson trichrome and elastin techniques, and vimentin immunohistochemistry. A coherent image stack was generated for three-dimensional image reconstructions, which were displayed using mixed rendering (i.e. differing graphics for separate tissue components) to show peri- and intra-bundle innervation against the muscle fascicle framework. RESULTS: Control specimens had a dense nerve supply. Muscle bundle innervation was derived by dichotomous branching from peri-bundle nerve trunks in the inter-bundle connective tissue. Transverse interfascicular branches entered bundles perpendicular to the long axis at the midpoint of the bundle. They gave rise to axial interfascicular branches, which distributed to the pre-terminal and terminal nerve fibres. All samples from patients with neurogenic DO had patchy denervation. The primary deficit was predominantly at the level of the terminal axial innervation and was cross-sectionally consistent along the longitudinal axis of the muscle bundle. CONCLUSION: Patchy denervation may reflect a deficit at the level of the peripheral ganglia. Any contraction in the areas of denervation either occurs out of co-ordination with the rest of the bladder, or is co-ordinated by means of non-neural structures. The observation of fine muscle strands running between fascicles, and connective tissue anchoring structures, represent two hypothetical mechanisms by which such co-ordination might be effected.

The sarcoplasmic reticulum in disease and smooth muscle dysfunction: therapeutic potential.

The functions of the sarcoplasmic reticulum (SR) in diseased smooth muscle can be investigated by measuring Ca2+ transients in response to agonist application, and through cell homogenization, isolation of microsomes and measurements of Ca-ATPase activity (SERCA). Such measurements have indicated that contractile dysfunction may be associated with degradation of SERCA in some systems, such as hypertrophied bladder smooth muscle. However, the postulated roles of the SR in smooth muscle function vary from one tissue to another and SR may mediate relaxation as well as contraction. Function seems to depend on the precise location of the SR with respect to the plasma membrane and its Ca2+-activated ion channels, the Ca2+ transporters, the cavaeoli, the mitochondria, and the contractile machinery. In diseases characterized by smooth muscle dysfunction, the size of the smooth muscle cells is frequently altered, as occurs in the hypertrophy seen in gut and bladder obstruction and hypertension. This will inevitably lead to alterations in the morphology and the function of the SR. Any therapeutic potential awaits considerable advances in our understanding of the systems in individual smooth muscles and the development of selective drugs.

Augmentation of nitric oxide to treat detrusor-external sphincter dyssynergia in spinal cord injury.

Detrusor-external sphincter dyssynergia (DSD) is a common cause of bladder outlet obstruction in men with spinal cord injuries, which if left untreated leads ultimately to renal failure. External sphincterotomy is currently the main treatment for DSD. However, obstruction persists in a substantial proportion of cases after this procedure. There is no effective drug treatment for DSD. Nitric oxide is an inhibitory neurotransmitter synthesised by nitric oxide synthase. Both animal and human studies suggest that nitric oxide mediates urethral sphincter relaxation. Nitric-oxide-synthase staining neurons have been identified in very high density in the urethral sphincters of a variety of animals and in human beings. Relaxation of the urethral sphincter is abolished by inhibitors of nitric oxide synthase and enhanced by nitric oxide donors. Mice with targeted deletion of the gene, for neuronal nitric oxide have urethral sphincters that do not relax in response to electrical stimulation. We hypothesise that augmentation of external sphincter nitric oxide could be an effective pharmacological treatment for DSD. Currently available nitric oxide donors such as glyceryl trinitrate or isosorbide mononitrate could be used to deliver nitric oxide to the urethral sphincter. The variable pharmacokinetics of these drugs combined with different modes of delivery (sublingual, buccal, or oral) could be used to achieve both short-term and long-term increases in concentrations of sphincter nitric oxide, thereby resulting in either acute or chronic lowering of urethral pressure. The safety and efficacy of this potential treatment for DSD needs to be established in clinical trials of men with spinal cord injures with DSD.

Morphological and physiological characteristics of urethral circular and longitudinal smooth muscle.

The urethral wall contains circular and longitudinal smooth muscle. Both layers can develop spontaneous tone, and contract further or relax in response to excitatory or inhibitory stimuli. In the pig the cells do not generate action potentials, and the membranes possess L-type calcium channels and a variety of potassium channels which may modulate the membrane potential and tone. The importance of these layers in generating the urethral pressure is not well understood in the human, but it seems likely that the circular smooth muscle is involved in generating urethral pressure in the pig.

The pharmacology of the internal anal sphincter and new treatments of ano-rectal disorders.

Surgical options for faecal incontinence in the presence of intact sphincters are limited. Furthermore, in patients with fissures, lateral sphincterotomy reduces anal sphincter hypertonia but there has been concern about complications. A greater understanding of the basic pharmacology of the internal anal sphincter has led to the development of novel treatments for both these disorders. A Medline review was undertaken for internal anal sphincter pharmacology, anal fissures and faecal incontinence. This review is based on these articles and those found by further cross-referencing. Nitric oxide released from non-adrenergic non-cholinergic nerves is the main inhibitory agent in the internal anal sphincter. Relaxations are also mediated through beta-adrenoceptors and muscarinic receptors. Stimulation of alpha-receptors results in contraction. Calcium and its entry through L-type calcium channels is important for the maintenance of tone. Nitric oxide donors produce reductions in resting anal tone and heal fissures but are associated with side-effects. Muscarinic agents and calcium channel antagonists show promise as low side-effect alternatives. Botulinum toxin appears more efficacious than other agents in healing fissures. To date, alpha-receptor agonists have been disappointing at improving incontinence. Further understanding of the pharmacology of the internal anal sphincter may permit the development of new agents to selectively target the tissue with greater efficacy and fewer side-effects.

Dual action of ZD6169, a novel K(+) channel opener, on ATP-sensitive K(+) channels in pig urethral myocytes.

1. The effects of ZD6169, a novel K(+) channel opener, on both membrane and unitary currents in pig urethra were investigated using patch-clamp techniques. Its effect was also examined on currents in inside-out patches of COS7 cells expressing carboxy terminus truncated inwardly rectifying K(+) channel (Kir6.2) subunits (Kir6.2C36) which form ATP-sensitive K(+) channels (K(ATP) channels). 2. In current-clamp mode, ZD6169 (< or = 10 microM) induced a concentration-dependent membrane hyperpolarization. Higher concentrations (> or = 30 microM) caused a transient membrane hyperpolarization, followed by a gradual membrane depolarization. On removal of ZD6169, an after hyperpolarization was observed. 3. In conventional voltage-clamp configuration, at -50 mV in symmetrical 140 mM K(+) conditions, ZD6169 (100 microM) caused a transient inward current which gradually decayed. Removal of ZD6169 evoked a much larger amplitude K(+) current with a similar time course. 4. ZD6169 produced an inward glibenclamide-sensitive K(+) current, demonstrating a bell-shaped concentration-response relationship. 5. In cell-attached configuration in symmetrical 140 mM K(+) conditions, ZD6169 (< or = 30 microM) activated an K(ATP) channel which was reversibly suppressed by application of glibenclamide. In contrast, ZD6169 (100 microM) inhibited the activity of the levcromakalim-induced K(ATP) channels. 6. ZD6169 (100 microM) had no significant effect on the channel activity of Kir6.2C36 in inside-out configuration, although cibenzoline greatly suppressed the channel activity. 7. These results demonstrate that ZD6169 possesses a dual effect on the activity of the K(ATP) channel; activating at low concentration and inhibiting at higher concentration.

Morphological aspects of the female pig bladder neck and urethra: quantitative analysis using computer assisted 3-dimensional reconstructions.

PURPOSE: To investigate whether the pig is a suitable model for studies of lower urinary tract function and dysfunction, we sought to determine the morphology of the female pig bladder neck and urethra. Computer assisted 3-dimensional (D) reconstructions from step serial histological sections were used for visualization of the spatial relationships between neighboring urethral wall components, and the quantification of these components in the bladder neck and along the urethra. MATERIALS AND METHODS: Step serial histological paraffin sections from the bladder neck and urethra of 6 female pigs, stained with Masson's trichrome, were used to generate computer assisted 3-D reconstructions using MacStereology (Ranfurly MicroSystems Ltd., Airdrie, United Kingdom) as the 3-D software package. RESULTS: The bladder neck and urethral anatomy revealed well defined smooth and striated muscle layers that varied in location, regional distribution and orientation. Circular smooth muscle was maximally developed in the mid urethra, at which point maximal urethral pressure was observed. The longitudinal smooth muscle layer appeared continuous with the detrusor, implicating a possible role in urethral shortening at the onset of voiding. A small circular and longitudinal striated muscle component was present in the distal urethra. CONCLUSIONS: Anatomical differences exist between the female pig and human bladder neck and urethra, which were successfully highlighted using computer assisted 3-D reconstructions from step serial histological paraffin sections.

The effect of bladder outflow obstruction on detrusor blood flow changes during the voiding cycle in conscious pigs.

PURPOSE: We monitored detrusor blood flow in pigs with bladder outflow obstruction. MATERIALS AND METHODS: Partial urethral obstruction was created in 9 immature, female large white pigs with an implanted ring, and 10 normal animals were used for comparison. Urodynamic parameters and detrusor blood flow were measured using chronically implanted access catheters and laser Doppler fibers. Repeated recordings were made from each animal while it was lightly sedated. RESULTS: The animals with implanted rings developed prolonged, high pressure voiding contractions in association with poor urinary flow, indicating bladder outflow obstruction, and had evidence of detrusor instability. In obstructed and normal animals detrusor blood flow was maintained during bladder filling. Elevated detrusor pressure during voiding significantly decreased blood flow to similar levels in each group. The duration of the ischemic period was much greater in obstructed animals. CONCLUSIONS: Bladder outflow obstruction is associated with repeated episodes of prolonged detrusor ischemia which may account for the biochemical and neuronal alterations in such bladders.