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Background: Interstitial cells of Cajal, expressing the proto-oncogene c-kit, have been shown to regulate the spontaneous activity of the gastrointestinal tract. They have been described in the human internal anal sphincter; however, their function is still unclear., Objective: We examined the effects of the c-kit tyrosine kinase inhibitor imatinib mesylate on sphincter strips to investigate the function of the interstitial cells., Design: This was a case series study., Settigs: This was a single-center study conducted at the University of Oxford., Patients: Internal anal sphincter strips were collected from 10 patients undergoing abdominoperineal resection or proctectomy and mounted in organ bath. Responses to electrical field stimulation and chemical agents were monitored in the absence of drugs and after the administration of increasing doses of imatinib mesylate. Immunohistochemistry was performed to identify interstitial cells., Main Outcome Measures: The role of the interstitial cells in the internal anal sphincter was assessed., Results: Imatinib mesylate significantly reduced the tone and the spontaneous activity of the strips. In the absence of drugs, the tone generated was 147.7 ± 33.0 mg/mg of tissue. Administration of ≥5 μM of imatinib mesylate caused a dose-dependent reduction in the tone. Strips exhibited spontaneous activity characterized by intermittent low-amplitude contractions superimposed on basal tone (135.6 ± 4.6 contractions in 10 minutes). Imatinib mesylate significantly reduced the number of contractions at concentration >5 μM. No differences were observed in the responses to electrical field stimulation, carbachol, or phenylephrine. Immunohistochemistry showed c-kit-positive cells., Limitations: This study was limited by the relatively small number of patients enrolled and thus the difficulty of finding human tissue for laboratory studies., Conclusions: Our results suggest that the interstitial cells modulate the tone and the spontaneous activity of the internal anal sphincter. This provides a foundation for new approaches to preclinical and clinical research. Moreover, these cells may represent a target for drugs inhibiting the c-kit receptor and provide a new approach for treating anorectal diseases.

Background: Neoadjuvant chemoradiotherapy is recommended in the treatment of locally advanced rectal cancer. Studies have suggested that chemoradiotherapy adversely affects anorectal function. However, the functional implication and the underlying neuromyogenic changes involved in radiation-induced damage are poorly understood., Objective: This study evaluated the functional changes following chemoradiotherapy on the internal anal sphincter., Design and Patients: This article describes an in vitro study on the internal anal sphincter collected from patients undergoing abdominoperineal resection or proctectomy. Five patients were treated by surgery alone (control group), and 6 received preoperative chemoradiotherapy (treatment group). Sphincter strips were mounted in organ bath, and the responses to electrical field stimulation and drugs were monitored., Settings: The study was performed at the University of Oxford., Main Outcome Measures: The end points of this study were to investigate whether chemoradiotherapy has any significant effects on internal anal sphincter function and, subsequently, to establish the type of injury induced., Results: Chemoradiotherapy strips developed similar tone, but significantly lower spontaneous activity (p = 0.001) than controls. Electrical field stimulation induced relaxation, followed by contraction. At 50 Hz, electrical field stimulation produced 25.6 ± 4.9% (mean ± SE) of maximum relaxation followed by a contraction of 5.5 ± 0.9% of basal tone in chemoradiotherapy strips i9n comparison with 47.0 ± 6.2% (p = 0.009) and 17.7 ± 4.0% (p = 0.007) in controls. Relaxation was significantly attenuated by N-nitro-L-arginine. Significant differences were found in responses to carbachol (p = 0.018) and phenylephrine (p = 0.022), but not to sodium nitroprusside., Limitations: This work was limited by the relatively small number of patients enrolled, because of the difficulty of finding human tissue for laboratory studies, and the lack of long-term results., Conclusions: Chemoradiotherapy significantly impairs internal anal sphincter function and intrinsic nerves seem more susceptible than smooth muscle. The exclusion of anal canal from the radiation field is recommended, when oncologically safe.

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., (Copyright © 2012 Wiley Periodicals, Inc.)

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.

Aims: Smooth muscles from the urethra and internal anal sphincter (IAS) play an essential role in the maintenance of urinary and fecal continence. Any damage in these muscles may cause serious problems. The aim of this study was to directly compare the contractile properties of pig urethra and IAS taken from the same animal., Methods: Smooth muscle strips of urethra and IAS dissected from the same pig were transferred to organ baths superfused with Krebs' solution, loaded with 1 g tension and equilibrated for 1 hr. Carbachol and phenylephrine response curves and EFS responses were elicited in the absence and presence of inhibitors., Results: Both tissues developed tone during the 1 hr equilibration period. Carbachol (3 × 10(-6)-10(-3) M) contracted urethra whilst relaxing IAS. Guanethidine (10(-6) M) inhibited the carbachol responses in both tissues. L-NOARG (10(-4) M) decreased carbachol responses in IAS, but not in urethra. Phenylephrine (3 × 10(-6)-10(-2) M) contracted both tissues. EFS (1-40 Hz) induced a contractile response in urethra which was decreased with guanethidine (10(-6) M) and further blocked by atropine (10(-6) M). In the presence of both, a relaxation response was observed that is sensitive to NOS inhibitors especially at low frequencies. EFS induced a relaxation followed by a contraction in IAS strips. This contraction was blocked by guanethidine but not by atropine, and the remaining relaxation at 20 Hz was decreased with L-NOARG and increased with L-arginine., Conclusions: There are differences between urethra and IAS in terms of muscarinic activation and neural innervation, relevant for pharmacotherapy., (© 2010 Wiley-Liss, Inc.)

Objective: To differentiate between the effects of parasympathetic and sensorimotor stimulation of isolated mouse and guinea-pig bladders in vitro by measuring the pressure increases to electrical field stimulation (EFS) and then comparing the effects of botulinum neurotoxin A (BoNT-A) applied either to the lumen or to the external bathing medium., Materials and Methods: Isolated mouse and guinea-pig bladders and detrusor strips were exposed to EFS in vitro before and after the addition of BoNT-A. The rationale of this method was that BoNT-A applied to the outside of the bladder would first affect the parasympathetic nerves before diffusing inwards to affect the sensorimotor innervation. BoNT-A applied intravesically would first reach the sensorimotor nerves and only later the parasympathetic nerves. Initial experiments on strips of detrusor were conducted to establish the correct dosage and application time of BoNT-A., Results: Contrary to our expectations, BoNT-A application failed to produce any significant effects on either the detrusor strips or whole bladders., Conclusions: Our experimental design failed to show any effect of BoNT-A on the contractility of detrusor muscle strips or whole bladders from mice and guinea-pigs. The reason for this is unclear, but may be related to tissue spending inadequate time incubated with BoNT-A under physiological conditions.

The implications of cholinergic and nitrergic transmissions in ascending and descending reflex motor pathways of recto-anal region in rat model were evaluated using: (i) electrical stimulation; (ii) triple organ bath; and (iii) morphological techniques. Electrical stimulation to anal canal induced simultaneous ascending contractile responses of longitudinal and circular muscles of proximal rectum, local contraction of anal canal or contraction followed by relaxation of internal anal sphincter when external sphincter was dissected off. The stimulation of proximal rectum elicited local contractions of both rectal layers and descending contractions of internal sphincter or anal canal. Tetrodotoxin (0.1 microM) prevented the electrically elicited events. The ascending excitatory responses and the local and ascending contractions of longitudinal muscle were more pronounced than those of circular muscle suggesting dominant role of ascending reflex pathways and of longitudinal muscle in rectal motor activity. Choline acetyltransferase (ChAT)-containing fibres and nicotinamide adenine dinucleotide phosphate (NADPH)-diaphorase-positive neurons were observed in myenteric ganglia of rectum and anal canal. NG-nitro-l-arginine (0.5mM) increased the contractile ascending and descending responses. During atropine (0.3 microM) treatment the ascending and descending contractions were suppressed but not abolished and a relaxation revealed in ascending response of circular muscle and in descending responses of internal anal sphincter and anal canal. The relaxation was decreased by NG-nitro-l-arginine and increased by l-arginine (0.5mM). The results suggest that cholinergic excitatory ascending and descending pathways and nitric oxide-dependent inhibitory ascending neurotransmission(s) to rectal circular muscle and inhibitory descending to internal anal sphincter and anal canal are involved in reflex circuitry controlling motor activity of recto-anal region.

Pharmacological studies have suggested the existence of ATP-sensitive K(+) (K(ATP)) channel as a therapeutic target in urinary bladders; however, electrical properties have not yet been shown. Patch-clamp techniques were applied to investigate the properties of K(ATP) channels in pig detrusor cells. In whole-cell configuration, levcromakalim, a K(ATP) channel opener, induced a long-lasting outward current in a concentration-dependent manner. The current-voltage curve of the levcromakalim-induced membrane current intersected at approximately -80 mV. This current was abolished by glibenclamide. Intracellular application of 0.1 mM GDP significantly enhanced the levcromakalim-induced membrane current, whereas cAMP did not. Furthermore, neurotransmitters related to cAMP signaling, such as calcitonin gene-related peptide, vasointestinal peptide, adenosine, and somatostatin, had little effect on the membrane current. In cell-attached configuration, levcromakalim activated K(+) channels with a unitary conductance of approximately 12 pS. When the patch configuration was changed to inside-out mode, the K(+) channel activity ran down. Subsequent application of 1 mM GDP reactivated the channels. The openings of the approximately 12 pS K(+) channels in the presence of 1 mM GDP was suppressed by ATP and glibenclamide. In reverse transcription-polymerase chain reaction, K(+) channel pore 6.1 and sulfonylurea receptor (SUR)2A were predominant in pig detrusor cells. The 12 pS K(+) channel activated by levcromakalim in pig detrusor smooth muscle cells is a K(ATP) channel. The predominant expression of SUR2A can account for the lack of effect of neurotransmitters related to cAMP.

Oscillatory inward membrane currents (I(oscil-in)) reflecting intracellular Ca(2+) ([Ca(2+)](i)) activity in detrusor cells, are thought to play an important role in producing tonic bladder contractions during micturition. The present patch clamp study revealed a new activation mechanism: sodium nitroprusside (SNP), a nitric oxide (NO) donor induced I(oscil-in) in a subpopulation of detrusor cells. The inhibitory effect of niflumic acid on SNP-induced I(oscil-in) suggests that Ca(2+)-activated Cl(-) channels are responsible for this current. In addition, SNP-induced I(oscil-in) required the cooperation of Ca(2+) influx through SK&F96365-sensitive channels and intracellular Ca(2+) release channels sensitive to ryanodine but insensitive to xestospongin C (XeC). This is also true for muscarinic agonist (carbachol: CCh)-induced I(oscil-in). However, 1H-[1,2,4]Oxadiazolo[4,3-a]quinoxalin-1-one (ODQ), a guanylyl cyclase inhibitor, suppressed SNP-induced I(oscil-in) but not CCh-induced I(oscil-in). The results suggest that a subpopulation of detrusor cells employ the NO/cGMP cascade to potentiate bladder contraction. Mechanisms underlying NO-induced I(oscil-in) are likely to contribute not only to the physiology but also to the pathophysiology of the lower urinary tract.

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., (Copyright 2008 Prous Science, S.A.U. or its licensors. All rights reserved.)

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.

Aims: To characterize the detrusor muscle of the mouse urinary bladder in order to understand more precisely spontaneous contractile behavior of this organ. This study examined the spontaneous electrical activity and Ca(2+) dynamics of the detrusor smooth muscle and investigated the role of the urothelium., Materials and Methods: Detrusor smooth muscle strips were isolated from mouse bladders. The urothelium was either kept intact or removed. Changes in membrane potential were recorded using sharp electrode intracellular recording. To image Ca(2+) dynamics, tissue strips were exposed to 10 microM Oregon Green 488 BAPTA-1 AM for 70 min, and then image series were acquired with a laser-scanning confocal microscope., Results: (1) Mouse detrusor smooth muscle cells (SMCs) generate nifedipine-sensitive spontaneous action potentials (sAPs) at a low frequency (1.3 +/- 0.9 min(-1), n = 11) in preparations with intact urothelium. This frequency increased when the urothelium was removed (7 +/- 8.3 min(-1), n = 17) (P < 0.05, Student's t test). (2) Frequent ATP-mediated spontaneous depolarizations were recorded in all cells. (3) The frequency of whole cell Ca(2+) flashes of detrusor smooth muscle cells was higher in preparations with the urothelium removed (median 1.2 min(-1), n = 7) than in urothelium denuded preparations (median 0.6 min(-1), n = 7) (P < 0.01, Mann-Whitney U-test)., Conclusions: Spontaneous activity of the mouse detrusor smooth muscles was characterized enabling future comparative work on gene knock-out strains. Evidence suggesting release of an inhibitory factor by the urothelium was apparent., (Copyright 2007 Wiley-Liss, Inc.)

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.

Objectives: Our purpose was to examine the impact of diabetes mellitus (DM) on vasoreactivity and endothelial function of radial artery (RA) grafts ex vivo., Background: The arteriopathy associated with DM may influence the surgeon's choice of conduits for revascularization. Arterial conduits and especially the RA are prone to vasospasm in the perioperative period., Methods: The study population consisted of 98 patients with coronary artery disease undergoing coronary artery bypass grafting by using RA grafts. The maximum contractions of RA segments induced by K+ (66 mmol/l) and clinically important vasoconstrictors such as adrenaline (5 x 10(-5) mol/l), angiotensin II (10(-6) mol/l), and prostaglandin F2alpha (PGF2alpha) (10(-6) mol/l) were recorded. Relaxation of RA rings to carbachol (10(-4) mol/l) was used as a measure of endothelial function. Multivariate analysis was then applied to determine the role of clinical characteristics on the vasomotor responses to these agents., Results: Vessels from patients with DM had greater contractions in response to adrenaline (p < 0.05), angiotensin (p < 0.05), and PGF2alpha (p < 0.01) compared with non-DM vessels, despite the similar vasoconstrictions induced by high K+ (p = NS). Diabetes mellitus was also associated with smaller vasorelaxations in response to carbachol (p < 0.001). In multivariate analysis, DM was an independent predictor of RA contractions in response to adrenaline (beta [SE] 3.085 [1.410], p = 0.031), angiotensin II (beta [SE] 3.838 [1.552], p = 0.015), and PGF2alpha (beta [SE] 4.609 [1.908], p = 0.018) but not K+ (p = NS). Diabetes mellitus was also independently associated with the vasorelaxations in response to carbachol (beta [SE] -15.645 [2.622], p = 0.0001)., Conclusions: Diabetes mellitus is associated with impaired endothelial function and greater contractions of RA grafts in response to all of the clinically relevant vasoconstrictors. These findings suggest that the RA of diabetic patients may be more prone to spasm in response to endogenous vasoconstrictors, an observation with important implications for surgeons' choice of conduits in this cohort of patients.

Intracellular Ca2+ stores in rat and guinea-pig bladders and taenia caecum were studied in beta-escin skinned smooth muscle strips. 30 min of skinning with 40 microM and 80 microM beta-escin were the best parameters found to obtain good calcium response curves (10(-7)-10(-4) M) in rat and guinea pig, respectively. Calmodulin (1 microM) increased the calcium contractions significantly. pCa 6 was used to load intracellular stores and application of carbachol (50 microM) in all tissues then only contracted the tissues in the presence of guanosine-5'-triphosphate (GTP; 100 microM). Inositol triphosphate (IP3; 50 microM), applied after pCa 6, contracted all tissues. Carbachol added after IP3 or heparin (1 mg/ml) no longer caused a contraction in any of them. In bladders, caffeine (30 mM) but not ryanodine (5 microM) prevented the subsequent carbachol contraction. A slowly rising contraction with carbachol was elicited after caffeine (30 mM) or ryanodine (5 microM) in the taenia and after ryanodine in the bladders. Caffeine (30 mM) suppressed the calcium response curves in all tissues. Procaine (30 mM) blocked the carbachol (50 microM) contractions in bladders but not in taenia. These results suggest that calcium induced calcium release (CICR) and IP3 induced calcium release (IICR) release calcium from a common store in bladder but two different compartments in taenia.

Objective: To examine the effects of a new selective beta3-adrenoceptor agonist, GW427353 on human detrusor function, as beta2- and beta3-adrenoceptors have been identified in the bladder, and can mediate detrusor relaxation, but beta3-adrenoceptors are less widely distributed and beta3-adrenoceptor agonists should have the therapeutic advantage of producing fewer treatment side-effects., Patients and Methods: 'Normal' human detrusor was retrieved from 12 patients (mean age 56 years) at cystectomy and from organ donors. Detrusor strips (4 x 1 x 1 mm) were mounted in superfused organ baths. Tone was induced with carbachol (5 x 10(-7)m) before applying either a nonselective beta-adrenoceptor agonist (isoprenaline) or GW427353 (with or without the beta3-adrenoceptor antagonist, SR59230A). In addition, the effect of GW427353 was tested on intrinsic nerve-evoked smooth muscle contraction over time. Effects on spontaneous activity were also recorded., Results: GW427353 produced significant relaxation at concentrations of >10(-7)m; isoprenaline produced a significant effect from 10(-6)m, but otherwise both agonists had similar effects. The addition of SR59230A (10(-7)m), produced partial inhibition of the GW427353 response. GW427353 at 10(-6)m significantly reduced spontaneous activity within 10 min of incubation, and at higher concentrations (>5 x 10(-6)m) inhibited detrusor contractions evoked by electrical field stimulation., Conclusion: Neuropathic bladder dysfunction is characterized by increased spontaneous activity and involuntary detrusor contractions, which can result in urinary frequency, urgency, nocturia and incontinence. The novel feature of GW427353 is the ability to suppress spontaneous activity and produce significant relaxation in human detrusor tissue at low concentrations, whilst also inhibiting evoked detrusor contractions at higher concentrations.

The isolated mouse whole bladder was used to study in vitro bladder overactivity evoked by intramural nerve sensitization with bradykinin, mimicking neurogenic bladder overactivity secondary to bladder inflammation. Intravesical pressure responses to intramural electrical stimulation of intramural nerves were measured under isovolumetric condition. Validation showed that carbachol produced a dose-response curve closely mirroring that observed in the isolated muscle strips and demonstrated the dual nature of electrically evoked neurotransmission, consisting of a cholinergic component largely mediated by M(3) receptors and a purinergic component mediated by P2X receptors. ATP generated a biphasic dose-response curve, suggesting that the P2X receptors may be heterogeneous in distribution. Characterization of bradykinin receptors showed bradykinin to be extremely potent in exciting the bladder, producing a dose-response curve with an EC(50) of 90 nM, and bradykinin also enhanced electrically evoked bladder contractions. These effects were inhibited by the B(2) receptor antagonist HOE 140 (d-Arg(0)-Arg(1)-Pro(2)-Hyp(3)-Gly(4)-Thi(5)-Ser(6)-d-Tic(7)-Oic(8)-Arg(9)) but not the B(1) receptor antagonist desArg(10) HOE 140 (H-d-Arf-Arg-Pro-Hyp-Gly-Thi-Ser-d-Tic-Oic-OH) and were also modulated by alpha,beta,methyleneATP. The isolated mouse whole bladder has proved a viable, robust model in which to demonstrate the pharmacological characteristic of the bladder and adds to the repertoire of in vitro tools for investigating potential therapeutic agents.

Objective: To assess the functional response of the urethral striated muscle to activation of its nerves, using a novel isolated organ-bath preparation., Materials and Methods: The urethra of the female guinea-pig was chosen as a suitable model for investigation, as it is functionally and structurally similar to the human urethra. Female Dunkin-Hartley guinea-pigs (400-500 g) were used; for the histochemical and immunohistochemical experiments, unfixed urethras were cryo-sectioned (14 microm thick) and were stained using established methods. For in vitro experiments, whole urethras were suspended vertically, with pudendal nerves intact, for isometric tension and intraluminal pressure recording in a 40-mL organ bath. Drugs were applied directly to the bathing solution., Results: In the striated muscle layer of the urethra there was positive beta-NADPH-diaphorase activity. In organ-bath studies the pudendal nerve-evoked contractions (0.2 ms pulses, 5 s trains, 70 V and 1-100 Hz) were abolished in the presence of tubocurarine (10(-6)m), and unaffected by guanethidine and atropine (both 10(-6)m). Pre-incubation with sodium nitroprusside and SIN-1 chloride significantly reduced the initial peak pressure responses (P < 0.05, anova for paired data) evoked by electrical field stimulation of the pudendal nerves at stimulus parameters of 0.2 ms pulses, 5 s trains, 70 V and 25 Hz., Conclusion: Electrically induced contractions were abolished by tubocurarine, confirming that the pudendal nerve innervates the striated muscle of the guinea-pig external urethral sphincter via nicotinic receptors. beta-NADPH-diaphorase histochemistry gave positive staining around guinea-pig striated muscle cells and possibly identified neuromuscular junction sites staining positively for the nitric oxide synthase marker. Together with the results of the organ-bath experiments, the results suggest that the striated muscle cells of the guinea-pig urethra have the machinery to respond to nitric oxide.

Unlabelled: OBJECTIVE To study the involvement of extracellular Ca2+ and the properties of the intracellular Ca2+ ([Ca2+]i) stores on the carbachol-induced contraction of mammalian urinary bladder smooth muscle strips under polarized and depolarized conditions., Materials and Methods: Strips of bladder were suspended between platinum ring electrodes in a cylindrical organ bath (0.2 mL) and continuously superfused with Krebs' solution at 1 mL/min. The effect of nifedipine, cyclopiazonic acid (CPA), thapsigargin, procaine, ryanodine and caffeine before and during a 10-s application of 100 microm carbachol under polarized conditions were studied. The effect of these drugs was also assessed under depolarized conditions using a protocol that allowed a more detailed assessment of the role of [Ca2+]i stores, consisting of emptying the stores by exposure to Ca2+-free solution, rapidly refilling them by a 10-s application of 81.5 mm Ca2+ (priming), returning to the Ca2+-free solution for 3 min and then applying 100 microm carbachol (10 s) in Ca2+-free solution (store release)., Results: Under polarized conditions, nifedipine and Ca2+ removal almost completely inhibited the carbachol-induced contractions. CPA increased the amplitude and duration of both carbachol- and electrical field stimulation-induced contractions. Although ryanodine had no inhibitory effect, caffeine and procaine significantly inhibited the carbachol-induced contraction. Under depolarized conditions nifedipine blocked both priming and store release contractions. CPA, thapsigargin, procaine and ryanodine significantly increased the priming and inhibited the store release contractions. However, caffeine virtually abolished both priming and store release contractions., Conclusion: These results suggest that in guinea-pig urinary bladder smooth muscle the Ca2+ necessary for contraction enters the cell through voltage-dependent dihydropyridine-sensitive Ca2+ channels and is pumped into an intracellular store that is released by carbachol. Under polarized conditions, the blockade of sarco-endoplasmic reticulum calcium ATP-ase (SERCA) with CPA increases [Ca2+]i and carbachol-induced contractions. The effects of caffeine and procaine suggest that store release involves ryanodine receptors and calcium-induced calcium release. Under depolarized conditions, Ca2+ entry is blocked by nifedipine and the stores diminish. Stored Ca2+ is also greatly reduced by the blockade of SERCA with either CPA or thapsigargin. Procaine, ryanodine and caffeine blocked the store release contractions, suggesting that this involves ryanodine receptors and calcium-induced calcium release.

1. The signal transduction pathways involved in carbachol (CCh)-induced calcium sensitization in beta-escin permeabilized rat and guinea-pig bladder smooth muscles were investigated and the results were compared with guinea-pig taenia caecum. 2. Calcium contractions elicited cumulatively (pCa 7.5-5) in the presence of calmodulin were significantly increased in all three tissues when CCh (50 microM) was added to the medium. 3. Under constant [Ca2+]i conditions (pCa 6), calmodulin (1 microM) and then GTP (100 microM) initiated significant contractions. CCh (50 microM) added to the bath caused a further contraction in all three tissues - calcium sensitization. This sensitization was significantly inhibited by atropine (50 microM). 4. The incubation of the tissues with the IP3-receptor blocker 2-APB (30 microM) reduced the subsequent development of calcium sensitization by CCh in rat bladder but did not affect it in guinea-pig bladder and taenia ceacum. 5. The Rho kinase (ROK) inhibitor Y-27632 (5 microM) added in the presence of CCh reversed the calcium sensitization in rat bladder, whereas a transient contraction followed by a relaxation to a level not significantly different from the CCh contraction was seen in both guinea-pig bladder and taenia caecum. Y-27632 (1 microM) continuously present significantly inhibited the CCh-induced Ca2+ sensitization in rat bladder but not in guinea-pig bladder or taenia caecum. 6. In the presence of cyclopiazonic acid (CPA) (1 microM) and calmodulin (1 microM), Y-27632 (5 microM) did not change the calcium response curve (3 x 10(-7)-10(-5) M) in rat bladder but increased the contractile responses significantly in both guinea-pig bladder and taenia caecum. 7. The protein kinase C (PKC) inhibitor GF 109203X (5 microM) added in the presence of CCh inhibited the calcium sensitization induced by this muscarinic agonist in all three tissues in different ratios. 8. In conclusion, muscarinic receptor activation induces calcium sensitization in rat and guinea-pig detrusor smooth muscles but there are differences in their pathways.

Objective: To describe the effect of a specific c-kit receptor inhibitor (imatinib mesylate) on human detrusor strips in vitro and guinea-pig cystometry in vivo, and to show histological data suggesting differences in the distribution of interstitial cells of Cajal (ICC)-like cells in 'normal' and overactive human detrusor, as these cells have been identified as possible mediators of spontaneous activity and excitability in bladder smooth muscle., Materials and Methods: Specimens of human detrusor were stained immunohistochemically with a c-kit antibody. Human detrusor strips were mounted in a superfused organ-bath apparatus, and smooth muscle contraction was evoked with carbachol and electrical field stimulation in the presence and absence of imatinib mesylate. Also, guinea-pig urodynamic studies were conducted before and after i.v. administration of imatinib mesylate, and changes in bladder variables and spontaneous activity were recorded., Results: Imatinib mesylate (10(-6)M) inhibited evoked smooth muscle contraction and spontaneous activity in overactive human detrusor, with less effect on normal human tissue. Imatinib mesylate (10(-5)M) improved bladder capacity, compliance, voided volumes, urinary frequency, and reduced contraction thresholds and spontaneous activity during guinea-pig cystometry. c-kit labelling showed significantly more ICC-like cells in overactive human detrusor than in normal specimens., Conclusion: c-kit receptor blockers have inhibitory effects on guinea-pig and overactive human detrusor, possibly via c-kit receptors on bladder ICC-like cells. This and the possibility that there are more ICC-like cells in overactive bladder suggest that the c-kit receptor may provide a novel target for treating detrusor overactivity.

The properties of smooth muscle are currently being studied extensively. Indeed, the small size of myocytes and the huge range of behaviours they exhibit make them an attractive focus for current research. However, this was not always the case. These properties initially made smooth muscles more difficult to study than the larger specialized striated muscles that were the focus of attention of leading researchers. In the UK, research into the physiology of smooth muscles began in the Pharmacology Department at Oxford, led by Edith Bülbring; her early results attracted much attention and resulted in the formation of an active international smooth muscle research group. Although several areas of current interest in the field of smooth muscle were not tackled by the Oxford group, progression of much of the field has clear links to Bülbring and her group.

Aims: The main focus of this study was to profile and compare urethral function in the female guinea pig and rat, and to characterize urodynamically distinctive patterns in the micturition cycle of the two species. This exercise aimed to investigate potential species-related differences and determine a suitable animal model for the human urethra., Materials & Methods: Female Dunkin-Hartley guinea pigs (400-500 g) and Sprague-Dawley rats (200-300 g) were used throughout the study. For in vitro experiments urethral rings were suspended vertically for isometric tension recording in 5 ml organ baths and drugs (phenylephrine 10(-4) M and carbachol 10(-4) M) were applied directly to the bathing solution. In vivo urethral pull-through and urodynamic studies were performed under urethane anesthesia, and phenylephrine (200 microg/kg) was administered during urethral pull-through experiments via the intravenous (i.v.) route. Urethral, bladder and arterial pressures, and external urethral sphincter electromyographic (EMG) spike activity were recorded simultaneously throughout., Results: Organ bath studies coupled with in vivo pull-through experiments produced urethral profiles with distinct regional variations for both species. Urodynamic studies with the urethral probe fixed in the high pressure zones of the proximal urethra produced similar recordings in both species during bladder filling and marked differences during micturition. The guinea pig showed complete sphincteric inhibition immediately prior and during micturition as also seen in the human, whereas the rat exhibited high frequency bursts in EMG activity at this stage in the cycle., Conclusions: The EMG activity seen in the rat during micturition is presumably necessary for efficient voiding, thus the guinea pig is a more suitable comparative model for the human in relation to urethral properties during micturition., (Neurourol. Urodynam. (c) 2005 Wiley-Liss, Inc.)

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.

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).

Aims: In the gastrointestinal tract, slow wave activity in smooth muscle is generated by the interstitial cells of Cajal (ICC). Detrusor smooth muscle strips of most species show spontaneous contractions which are triggered by action potential bursts, however, the pacemaker mechanisms for the detrusor are still unknown. Recently, ICC-like cells have been found in guinea-pig bladder, using antibodies to the c-kit receptor. We have investigated the effects of Glivec, a c-kit tyrosine kinase inhibitor, on spontaneous action potentials in guinea-pig detrusor and intravesical pressure of isolated guinea-pig bladders., Methods: Changes in the membrane potential were measured in guinea-pig detrusor smooth muscle using conventional microelectrode techniques. Pressure changes in the bladder were recorded using whole organ bath techniques., Results: Smooth muscle cells in detrusor muscle bundles exhibited spontaneous action potentials, and spontaneous pressure rises occurred in isolated bladders. Glivec (10 microM) converted action potential bursts into continuous firing with no effects on the shape of individual action potentials. Glivec (>50 microM) reduced the amplitude of spontaneous pressure rises in the whole bladder in a dose dependent manner and abolished spontaneous action potentials in detrusor smooth muscle cells., Conclusions: The results suggest that ICC-like cells may be responsible for generating bursts of action potentials and contractions in detrusor smooth muscle. Drugs inhibiting the c-kit receptor may prove useful for treating the overactive bladder.

Scientists interested in the smooth muscles of the urinary tract, and their control, have recently been studying cells in the interstitium of tissues that express the c-kit antigen (Kit(+) cells). These cells have morphologic features that are reminiscent of the well-described pacemaker cells in the gut, the interstitial cells of Cajal (ICC). The spontaneous contractile behavior of muscles in the urinary tract varies widely, and it is clear that urinary tract Kit(+) interstitial cells cannot be playing an identical role to that played by the ICC in the gut. Nevertheless, there is increasing evidence that they do play a role in modulating the contractile behavior of adjacent smooth muscle, and might also be involved in mediating neural control. This review outlines the properties of ICC in the gut, and gives an account of the discovery of cells in the interstitium of the main components of the urinary tract. The physiologic properties of such cells and the functional implications of their presence are discussed, with particular reference to the bladder. In this organ, Kit(+) cells are found under the lamina propria, where they might interact with the urothelium and with sensory nerves, and also between and within the smooth-muscle bundles. Confocal microscopy and calcium imaging are being used to assess the physiology of ICC and their interactions with smooth muscles. Differences in the numbers of ICC are seen in smooth muscle specimens obtained from patients with various pathologies; in particular, bladder overactivity is associated with increased numbers of these cells.

Inhibitory innervation of urethral smooth muscle is mediated partly through release of NO. We investigated the mechanisms involved in the supply of the substrate L: -arginine to NO synthase by examining the relaxant response of the muscle to electrical field stimulation (EFS) and the effects of addition of amino acids to the bathing medium. Relaxant responses persisted during hours of repetitive stimulation but were enhanced rapidly by addition of L: -arginine (the "arginine paradox"). Addition of L: -lysine (competes with L: -arginine for transport on the y(+) carrier) and L: -glutamine (competing on the y(+)L carrier) attenuated the enhancement. Enhancement persisted after washing but was reversed by application of L: -lysine, suggesting that exogenous L: -arginine fills an intracellular pool and that L: -lysine can trans-stimulate its efflux from the pool. After prolonged depolarization in high-K(+), Na(+)-free solution the relaxant response became purely nitrergic. Addition of L: -arginine during the exposure continued to enhance the subsequent responses but L: -glutamine added with L: -arginine, could no longer reduce this enhancement. The results show the arginine paradox in inhibitory nerves and suggest the involvement of y(+) and y(+)L carriers in the transport of L: -arginine.

The urinary bladder pressure during micturition consists of two components: an initial, phasic component and a subsequent, sustained component. To investigate the excitation mechanisms underlying the sustained pressure, we recorded from membranes of isolated detrusor cells from the pig, which can be used as a model for human micturition. Parasympathomimetic agents promptly evoke a large transient inward current, and subsequently during its continuous presence, oscillating inward currents of relatively small amplitudes are observed. The two types of inward current are considered to cause the phasic and sustained pressure rises, respectively. Ionic substitution and applications of channel blockers revealed that Ca(2+)-activated Cl(-) channels were responsible for the large transient and oscillating inward currents. Furthermore, the inclusion of guanosine 5'-O-(2-thiodiphosphate) in the patch pipette indicates that both inward currents involve G proteins. However, applications of heparin in the patch pipette and of xestospongin C in the bathing solution suggest a signaling pathway other than inositol 1,4,5-trisphosphate (IP(3)) operating in the inward current oscillations, unlike the initial transient inward current. This IP(3)-independent inward current oscillation system required both sustained Ca(2+) influx from the extracellular space and Ca(2+) release from the intracellular stores. These two requirements are presumably SKF-96365-sensitive cation channels and ryanodine receptors, respectively. Experiments with various Ca(2+) concentrations suggested that Ca(2+) influx from the extracellular space plays a major role in pacing the oscillatory rhythm. The fact that distinct mechanisms underlie the two types of inward current may help in development of clinical treatments of, for example, urinary incontinence and residual urine volume control.

Kinetic studies of U-37883A (4-morpholinecarboximidine-N-1-adamantyl-N'-cyclohexyl-hydrochloride), a vascular ATP-sensitive K+ channel (KATP channel) blocker, were performed on pig urethral myocytes to investigate inhibitory effects on large-conductance intracellular Ca2+ -sensitive K+ channels (i.e., BKCa channels; 225 pS K+ channels) by use of single-channel recordings (outside-out and inside-out configuration). BKCa channels in pig urethral smooth muscles showed extracellular iberiotoxin (300 nM) sensitivity and voltage dependency. The alpha subunit of BKCa channel proteins was detected in the membrane fraction by use of Western blot technique. Application of U-37883A (> or =10 microM) reduced the activity of BKCa channels in a concentration-dependent manner, not only by decreasing mean openlife time but also by prolonging the mean closed time. These results shows that U-37883A affects channels other than the vascular KATP channel, and demonstrates how it inhibits the activities of BKCa channels in urethral smooth muscles.

In order to assess the possible role of the c-kit positive cells in the bladder, the effects of c-kit tyrosine kinase inhibitor, Glivec, on spontaneous excitation and ion channel activity in detrusor smooth muscles of the guinea-pig bladder were investigated using intracellular microelectrodes, isometric muscle tension recordings and patch clamp techniques. Glivec (10 microM) converted action potential bursts into continuous firing without affecting their shape but at 50 microM abolished spontaneous action potentials. It had little effect on inward and outward currents at <10 microM, but inhibited them at >50 microM. Glivec decreased the amplitude of spontaneous contractions dose dependently. These results suggest that c-kit positive cells may play a role in modulating spontaneous electrical and mechanical activities. Drugs inhibiting the c-kit receptor may provide a new approach for treating the overactive bladder.

In the present investigation, we used standard patch clamp techniques to test whether nitric oxide (NO) generation has any role to play with either activation or inhibition of ATP-sensitive (KATP) channels in guinea-pig urinary bladder. We found that NO generation leads to activation of KATP channels through a cyclic guanosine monophosphate (c-GMP)-dependent protein kinase. 3-Morpholinosydnonimine (SIN, 100 microM) potentiated activation of an inward current in whole cell patch clamp experiments. Glibenclamide (10 microM) and 1H-[1,2,4]oxadiazolo[4,3-a]quinoxalin-1-one (ODQ, 10 microM) inhibited the SIN-activated current. Both in cell-attached and in inside out patches, SIN (200 microM) potentiated KATP channel activity, and the increased channel activity in inside out patches was suppressed by glibenclamide (50 microM), ATP (1 mM) and (9s,10R,12R)-2,3,9,10,11,12-Hexahydro-10-methoxy-2,9-dimethyl-1-oxo-9,12,-epoxy-1H-diindolo[1,2,3-fg:3',2',1'-kl]pyrrolo[3,4-i][1,6] benzodiazocine-10-carboxylic acid, methyl ester (KT-5823, 10 nM). 8-Br-cGMP (100 microM) increased the KATP channel activity in cell-attached patches, and this was suppressed by glibenclamide (50 microM). These results suggest that the NO-c-GMP-PKG pathway contributes to activation of K(ATP) channels in guinea-pig urinary bladder myocytes.

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., (Copyright 2003 British Journal of Surgery Society Ltd. Published by John Wiley & Sons, Ltd.)

1. To investigate the cellular mechanisms underlying spontaneous excitation of smooth muscle of the guinea-pig urinary bladder, isometric tension was measured in muscle bundles while recording the membrane potential from a cell in the bundle with a microeletrode. Changes in the intracellular calcium concentration ([Ca(2+)](i); calcium transients) were recorded in strips loaded with the fluorescent dye, fura-PE3. 2. In 40% of preparations, individual action potentials and contractions, which were abolished by nifedipine (1 microm), were generated. In the remaining preparations, bursting action potentials and contractions were generated. Contractions were again abolished by nifedipine (1 microm), while higher concentrations of nifedipine (10-30 microm) were required to prevent the electrical activity. 3. Carbachol (0.1 microm) increased the frequency of action potentials and corresponding contractions. Apamin (0.1 microm) potentiated bursting activity and enhanced phasic contraction. Charybdotoxin (CTX, 50 nm) induced prolonged action potentials that generated enlarged contractions. In contrast, levcromakalim (0.1 microm) reduced the frequency of action potentials, action potential bursts and the size of the contractions. 4. Forskolin (0.1 microm), 8-bromoguanosin 3', 5' cyclic monophosphate (8Br-cGMP, 0.1 mm) and Y-26763 (10 microm) suppressed contractions without reducing the amplitude of either action potentials or Ca transients. 5. This paper confirms that action potentials and associated calcium transients are fundamental mechanisms in generating spontaneous contractions in smooth muscles of the guinea-pig bladder. However, in parallel with the excitation-contraction coupling, the sensitivity of the contractile proteins for Ca(2+) may play an important role in regulating spontaneous excitation and can be modulated by cyclic nucleotides and Rho kinase.

Endothelin-1 (ET-1) is a powerful vasoconstricting peptide. Recent studies showed synthesis of ET-1 and the presence of ET receptors in urinary bladder smooth muscle cells. In the present study, we investigated the possible role of ET-1 in detrusor contraction and its underlying mechanisms in terms of electrical activity. ET-1 caused dose-dependent tonic contraction of bladder smooth muscle strips. Whole cell patch-clamp experiments revealed that ET-1 induced a single transient inward current in the majority of detrusor cells and that additional inward current oscillations were induced in one-third of the cells. The inward current oscillation and tonic contraction shared several characteristic features: 1) both activities lasted for a considerable time after ET-1 washout and 2) only prior application of ETA receptor antagonists, not ETB receptor antagonists, significantly suppressed ET-1-induced contractions and the oscillating inward currents. It was concluded that the inward current oscillation underlies ET-1-induced tonic contraction. Experiments with ion substitution and channel blockers suggested that periodic activation of Ca2+-activated Cl- channels caused the oscillating inward currents.

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.

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.

Purpose of Review: To assess the potential role of nerve regeneration in restoring urinary tract function, the rapidly developing and exciting area of central and peripheral nerve repair and regeneration is reviewed, with particular reference to papers in which animal models of nerve damage resulting in urogenital dysfunction have been used. The difficulties and potential of these techniques for therapeutic application to human subjects with functional problems of the urinary tract are discussed., Recent Findings: Methods for encouraging regeneration of cut axons and directed growth in the inhibitory environment of the central nervous system are being extensively explored. The recent discovery of the potential of olfactory ensheathing cells has proved a significant advance. Olfactory ensheathing cells are a type of glial cell which can be harvested from the olfactory mucosa. Transplantation of these cells, in conjunction with a biodegradable synthetic nerve guide or conduit, has been shown to restore urinary tract function after spinal cord injury. Artificial, biodegradable conduits have also restored bladder and spermatic duct function after sympathetic nerve damage. Other adjuvants facilitating the process of axonal recovery include the use of neurotrophins to accelerate and guide the formation of new nerve-fibre growth., Summary: These revolutionary technologies may, in the future, provide a means of treating urinary tract dysfunction with some types of aetiology, including acute spinal cord injury, and injury to nerves following pelvic surgery. It is, however, less likely that these treatments will be used successfully in the near future in patients in which the neural damage is long term, or associated with death of post-ganglionic neurons.

(1) The electrophysiological properties of detrusor smooth muscles have been studied almost exclusively in small mammals and the relevance of the information to the human bladder has been questioned. In the present study, electrical properties of detrusor smooth muscles of the pig and human were investigated using intracellular recording techniques. (2) Bladder smooth muscles of the pig and human exhibited nifedipine (10 microm)-sensitive spontaneous action potentials, and their frequency was highly sensitive to membrane polarization. (3) During bursts of action potentials, each action potential was followed by a fast after-hyperpolarization (fast AHP). Charybdotoxin (CTX, 50 nm) increased the amplitude and duration of action potentials but failed to inhibit the fast AHPs, while apamin (0.1 microm) blocked the fast AHPs and induced action potential complexes, which were followed by slow AHPs. 4-Aminopyridine (4-AP, 1 mm) suppressed the slow AHP and increased action potential frequency. (4) In the human bladder, transmural stimuli initiated inhibitory junction potential-like hyperpolarizations, which were followed by action potential discharges. The hyperpolarizations were blocked by atropine (1 microm) and by apamin (0.1 microm) but not by CTX (50 nm). In the pig bladder, transmural stimuli evoked excitatory junction potentials (EJPs), which triggered action potentials. After desensitizing P2x receptors with alpha,beta methylene-ATP (10 microm), nerve-evoked responses were similar to those of human bladder. (5) These results indicate that detrusor smooth muscles of the pig share many features of electrical properties with those of the human. In addition to large conductance (BK) and small conductance (SK) Ca2+-activated K+ channels, voltage-dependent K+ (VK) channels may play an important role in the regulation of electrical activity of detrusor smooth muscles.

(1) The regulatory mechanisms of spontaneous excitation in detrusor smooth muscles of the guinea-pig urinary bladder were investigated using intracellular microelectrode and muscle tension recording techniques. (2) Detrusor smooth muscle cells exhibited nifedipine-sensitive spontaneous action potentials. Their frequency was highly sensitive to membrane polarization and was reduced by lowering the temperature. Lowering the temperature also reduced the frequency of spontaneous contractions and increased their amplitude. (3) Charybdotoxin (50 nm) and iberiotoxin (0.1 microm) increased the amplitude and duration of action potentials, and abolished after hyperpolarizations (AHPs). Both agents also increased the amplitude and duration of spontaneous contractions, and reduced their frequency. Apamin (0.1 microm) did not change the shape of action potentials but often converted individual action potentials into bursts. It also increased the amplitude and duration of spontaneous contractions, and reduced their frequency. 4-aminopyrideine (4-AP, 1 mm) increased the frequency of action potentials without affecting their shape, and increased the amplitude and frequency of spontaneous contractions. (4) Cyclopiazonic acid (CPA, 10 microm) and ryanodine (50 microm) increased the amplitude of action potentials, and suppressed AHPs. Both agents also increased the amplitude and duration of spontaneous contractions, and reduced their frequency. 1,2-(Bis (2-aminophenoxy) ethane-N,N,N', N'-tetraacetic acid tetrakis (acetoxymethyl ester) (50 microm) dramatically increased the amplitude and duration of the action potential, and abolished AHPs. (5) Spontaneous action potentials in detrusor smooth muscles cells result from the opening of L-type Ca2+ channels, and their frequency is regulated by voltage-dependent mechanisms and by some metabolic process. Both the activation of large conductance Ca2+-activated K+ (BK) channels and Ca2+-mediated inactivation of the Ca2+ channels are involved in the repolarizing phase of action potentials. The Ca2+ influx through L-type Ca2+ channels triggers calcium-induced calcium release via ryanodine receptors and activates BK channels to generate AHPs. Both small conductance Ca2+-activated K+ channels and voltage-sensitive K+ channels may contribute to the resting membrane potential and regulate the frequency of action potentials. The regulatory mechanisms of action potentials are closely related to the regulation of spontaneous contractions.

Purpose: In vitro data suggest that nitric oxide is an important inhibitory neurotransmitter in the internal anal sphincter, and morphologic evidence implies that it mediates the rectoanal inhibitory reflex. This study examined the anatomy, physiology, and pharmacology of the internal sphincter in control and neuronal nitric oxide synthase knockout mice., Methods: Neuronal nitric oxide synthase, nicotinamide adenosine triphosphate dinucleotide phosphate diaphorase histochemistry, and PGP 9.5 immunohistochemistry were compared between knockout and sibling control mice. Anorectal manometry was performed with a balloon-tipped water-perfused catheter. In vitro studies were performed on both whole internal anal sphincter rings and strips., Results: Staining of the myenteric plexus and nerves traversing the internal anal sphincter in sibling control mice demonstrated the presence of neuronal nitric oxide synthase and nicotinamide adenine dinucleotide phosphate diaphorase at these sites. These markers were absent in knockout mice. Maximum anal resting pressure was similar in control and knockout mice (15.6 +/- 2.6 cm H(2)O (n = 4) vs. 14.0 +/- 2.3 cm H(2)O (n = 7)). The rectoanal inhibitory reflex was present in all control mice (n = 4) but in only four of seven knockout mice. Field stimulation with parameters designed to activate inhibitory nerves produced relaxation of internal sphincter tissue from both control and knockout mice, which was partially attenuated in control mice only by pretreatment with the nitric oxide synthase inhibitor N omega-nitro-L-arginine. Further inhibition of nerve-induced relaxation in control mice was achieved with antagonists of vasoactive intestinal peptide, adenosine triphosphate, and heme oxygenase., Conclusions: Although in the normal mouse, nitric oxide is an inhibitory neurotransmitter in the internal sphincter, other transmitters may play a role in the rectoanal inhibitory reflex. These other inhibitory neurotransmitters can apparently compensate for the absence of nitric oxide synthase in knockout mice to maintain approximately normal function.

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., (Copyright 2003 British Journal of Surgery Society Ltd. Published by John Wiley & Sons, Ltd.)