ATP during early bladder stretch is important for urgency in detrusor overactivity patients.

ATP is an important mediator of urgency in women with detrusor overactivity (DO). In order to understand how different degrees of bladder stretch elicited ATP release in DO patients compared with controls, sequential aliquots were collected during cystometry and ATP release was measured at each degree of bladder filling, in female patients with DO and controls. In both DO and control groups, ATP release was induced during bladder filling, suggesting that stretch stimulated further ATP release. However, the luminal ATP concentrations were already high at early filling stage (200 mL), which was even greater than those at the later filling stages (400 mL and maximum cystometric capacity, MCC), indicating that a substantial ATP release has been induced during early filling (200 mL) in both DO and controls. In DO, ATP release at 200 mL was significantly higher in those with low first desire to void (FDV) (≤ 200 mL) than in those with higher FDV (> 200 mL); this may suggest that ATP release at early stretch may play an important role in urgency (early sensation) in DO. ATP concentrations remained unchanged after voiding, suggesting that voiding did not further induce ATP release into intraluminal fluid.

Alterations in acetylcholine, PGE2 and IL6 release from urothelial cells following treatment with pyocyanin and lipopolysaccharide.

The effects of pseudomonal virulence factor pyocyanin, and LPS from Pseudomonas aeruginosa and Escherichia coli on urothelial mediator release and cytokine production were examined. RT4 urothelial cells were treated with pyocyanin (1-100 µM) or LPS (1-100 ng/mL) for 24-h. Effects were measured in terms of changes in cell viability, basal and stretch-induced acetylcholine (Ach) and PGE2 release, and inflammatory cytokines (IL-6 and IL-12) production. Twenty-four hour pyocyanin (100 µM) treatment significantly decreased urothelial cell viability, while stretch-induced Ach release response was inhibited. E. coli LPS (100 ng/mL) produced a similar response with an additional significant increase in basal Ach release. All three virulence factors significantly increased urothelial PGE2 release; under basal release for pyocyanin (100 µM), stretch-induced release for pseudomonal LPS (≥ 10 ng/mL) and both basal and stimulated release for E. coli LPS (≥ 10 ng/mL). IL-6 and IL-12 were not detected in control samples, however 24h treatment with pyocyanin (100 µM) or LPS (100 ng/mL) resulted in IL-6 release from urothelial cells. The changes in urothelial Ach and PGE2, and release of inflammatory cytokine IL-6 induced by exposure to the bacterial virulence factors may play a role in the symptoms of pain and urinary urgency experienced with urinary tract infections.

Non-adrenergic, non-cholinergic, non-purinergic contractions of the urothelium/lamina propria of the pig bladder.

Acetylcholine, and to a lesser extent ATP, mediates neurogenic contractions of bladder smooth muscle. Recently, the urothelium and lamina propria have also been shown to have contractile properties, but the neurotransmitters involved in mediating responses to nerve stimulation have not been investigated. Isolated strips of porcine urothelium with lamina propria were electrically field stimulated and contractions recorded. Drugs interfering with neurotransmission were then employed to identify which neurotransmitters mediated responses. Strips of urothelium/lamina propria developed spontaneous contractions with a frequency of 3.5±0.1 cycles min⁻¹ and amplitude of 0.84±0.06 g. Electrical field stimulation at 5, 10, and 20 Hz resulted in frequency-related contractions (1.13±0.36 g, 1.59±0.46 g and 2.20±0.53 g, respectively, n=13), and these were reduced in the presence of tetrodotoxin (1 µm) by 77±20% at 5 Hz, 79±7% at 10 Hz and 74±12% at 20 Hz (all P<0.01), indicating they were predominantly neurogenic in nature. Neither the muscarinic antagonist atropine (10 µm), the adrenergic neurone blocker guanethidine (10 µm) nor desensitization of the purinergic receptors with α,ß-methylene ATP (10 µm) affected the contractile amplitude. Similarly, responses were not affected by the nitric oxide synthase inhibitor L-NNA (100 µm) or drugs that interfere with peptide neurotransmission (capsaicin, NK2 antagonist GR159897, protease inhibitors). In conclusion, electrical depolarization of the nerves present in the porcine urothelium/lamina propria results in frequency-dependent contractions, which are predominantly neurogenic in nature. These contractions are resistant to drugs that inhibit the adrenergic, cholinergic and purinergic systems. The neurotransmitter involved in the responses of this tissue is therefore unknown but does not appear to be a peptide.

Pharmacology of the internal anal sphincter and its relevance to faecal incontinence.

1 The internal anal sphincter (IAS) has a spontaneous tone and is the main contributor to the maintenance of faecal continence. The spontaneous resting tone exhibited by the sphincter can be modified by neurotransmitters from the autonomic and enteric nervous systems. 2 In this review, the influence of the sympathetic and parasympathetic nervous systems on IAS tone are discussed and the putative roles of nitric oxide, carbon monoxide, vasoactive intestinal peptide and adenosine triphosphate in non-adrenergic non-cholinergic transmission are considered. 3 Faecal incontinence is a common condition that places a heavy financial burden on the health service and severely affects patients' quality of life. Resting anal pressure is reduced in patients with faecal incontinence and agents that increase sphincter tone tend to relieve symptoms. The results of clinical studies of the use of phenylephrine to treat faecal incontinence are reviewed. 4 It is concluded that the IAS is a potential target for drug development for the treatment of faecal incontinence.

Bladder afferent sensitivity in wild-type and TRPV1 knockout mice.

Understanding bladder afferent pathways may reveal novel targets for therapy of lower urinary tract disorders such as overactive bladder syndrome and cystitis. Several potential candidate molecules have been postulated as playing a significant role in bladder function. One such candidate is the transient receptor potential vanilloid 1 (TRPV1) ion channel. Mice lacking the TRPV1 channel have altered micturition thresholds suggesting that TRPV1 channels may play a role in the detection of bladder filling. The aim of this study was therefore to investigate the role of TRPV1 receptors in controlling bladder afferent sensitivity in the mouse using pharmacological receptor blockade and genetic deletion of the channel. Multiunit afferent activity was recorded in vitro from bladder afferents taken from wild-type (TRPV+/+) mice and knockout (TRPV1-/-) mice. In wild-type preparations, ramp distension of the bladder to a maximal pressure of 40 mmHg produced a graded increase in afferent activity. Bath application of the TRPV1 antagonist capsazepine (10 mum) caused a significant attenuation of afferent discharge in TRPV1+/+ mice. Afferent responses to distension were significantly attenuated in TRPV1-/- mice in which sensitivity to intravesical hydrochloric acid (50 mm) and capsaicin (10 microm) were also blunted. Altered mechanosensitivity occurred in the absence of any changes in the pressure-volume relationship during filling indicating that this was not secondary to a change in bladder compliance. Single-unit analysis was used to classify individual afferents into low-threshold and high-threshold fibres. Low threshold afferent responses were attenuated in TRPV1-/- mice compared to the TRPV1+/+ littermates while surprisingly high threshold afferent sensitivity was unchanged. While TRPV1 channels are not considered to be mechanically gated, the present study demonstrates a clear role for TRPV1 in the excitability of particularly low threshold bladder afferents. This suggests that TRPV1 may play an important role in normal bladder function.

Investigation of neurokinin-2 and -3 receptors in the human and pig bladder.

OBJECTIVE: To investigate the role of neurokinin (NK)-2 and -3 receptors in mediating the contraction of detrusor muscle strips from human and pig, to determine whether the pig is a good model for the study of tachykinin receptors in the human bladder, as the biological actions of tachykinins, e.g. substance P and NKA are mediated via three distinct receptor subtypes, NK-1, -2 and -3. MATERIALS AND METHODS: Strips of detrusor muscle were obtained from the bladder dome and neck of female pigs and from patients undergoing cystectomy. Cumulative concentration-response curves to NKA were obtained in the absence and presence of either the NK-2 receptor-selective antagonist SR48968 or the NK-3 receptor-selective antagonist SB223412. RESULTS: NKA produced concentration-dependent contractions in the human and pig detrusor muscle; the curves were shifted to the right by SR48968, with high affinity (pKB 8.9, 8.3 and 8.0 in the human, pig dome and pig neck, respectively), whereas SB223412 had a minimal effect (pKB 5.8, 5.8 and 6.3, respectively). CONCLUSION: These data confirm that the NK-2 receptor subtype mediates NKA-induced contraction of the human and pig detrusor muscle. The NK-3 receptor appears to have no role in detrusor contraction of either species. The results also provide evidence that the NK-2 receptor in human and pig are the same, and the latter may be an appropriate species to study tachykinin-induced contractions in human bladder.

Muscarinic receptors of the urinary bladder: detrusor, urothelial and prejunctional.

1. The parasympathetic nervous system is responsible for maintaining normal bladder function, contracting the bladder smooth muscle (detrusor) and relaxing the bladder outlet during micturition. 2. Contraction of the bladder involves direct contraction via M3 receptors and an indirect 're-contraction' via M2-receptors whereby a reduction in adenylate cyclase activity reverses the relaxation induced by beta-adrenoceptor stimulation. 3. Muscarinic receptors are also located on the epithelial lining of the bladder (urothelium) where they induce the release of a diffusible factor responsible for inhibiting contraction of the underlying detrusor smooth muscle. The factor remains unidentified but is not nitric oxide, a cyclooxygenase product or adenosine triphosphate. 4. Finally, muscarinic receptors are also located prejunctionally in the bladder on cholinergic and adrenergic nerve terminals, where M1-receptors facilitate transmitter release and M2 or M4-receptors inhibit transmitter release. 5. In pathological states, changes may occur in these receptor systems resulting in bladder dysfunction. Muscarinic receptor antagonists are the main therapeutic agents available for treatment of the overactive bladder, but whether their therapeutic effect involves actions at all three locations (detrusor, prejunctional, urothelial) has yet to be established.

Active relaxation of human gallbladder muscle is mediated by ATP-sensitive potassium channels.

BACKGROUND: Active and significant relaxation of the human gallbladder must be one of the facets of its motility during both the filling and emptying cycle. Conflicting reports about the presence or significance of nitric oxide have been reported in the literature. The aim of this study was to investigate the role of nitric oxide and K(ATP) channels in human gallbladder muscle using isolated strips from human gallbladder. METHODS: Full thickness strips were obtained from 56 human gallbladders and suspended under isometric tension in organ baths. The effect of nitric oxide donors and inhibitors on cholecystokinin octapeptide- and carbachol-induced contraction was examined. In separate experiments the effect of the K(ATP) channel activator, cromakalim, and the inhibitor, glibenclamide, were determined. RESULTS: Cromakalim induced a significant relaxation of agonist-induced contraction in human gallbladder in vitro, an effect which was abolished by the K(ATP) channel inhibitor glibenclamide. No evidence of significant nitric oxide involvement in relaxation was observed. CONCLUSIONS: This study has demonstrated the presence of K(ATP) channels in human gallbladder for the first time. These are capable of causing significant relaxation in the presence of hormonal and muscarinic agonists and may represent a major pathway for gallbladder relaxation.

The effects of streptozotocin-induced diabetes and aldose reductase inhibition with sorbinil, on left and right atrial function in the rat.

Diabetes mellitus is frequently associated with the complications of cardiovascular disease. Activation of the aldose reductase (or polyol) pathway has long been implicated as an underlying factor for the development of many diabetic complications and indeed, treatment with aldose reductase inhibitors has been shown to prevent or reverse many of these diabetic complications. This study examines the effects of 14-day streptozotocin-induced diabetes on alpha1- and beta-adrenoceptor-mediated responses in rat isolated left and right atria. The effects of treatment with the aldose reductase inhibitor (ARI) sorbinil were also studied. A positive inotropic response was observed to both isoprenaline and phenylephrine in left atria. Diabetes of 14 days duration resulted in a supersensitivity of these tissues to the beta-adrenoceptor agonist in comparison with controls, while responses to the alpha1-adrenoceptor agonist were unaltered. Spontaneously beating right atria from diabetic rats was found to have a depressed resting rate compared with control tissues, although positive chronotropic beta-adrenoceptor-mediated responses were not affected by diabetes. Phenylephrine produced alpha1-adrenoceptor-mediated chronotropic responses in right atrial tissues, and these were found to be enhanced in rats with diabetes. Treatment of diabetic rats with the ARI sorbinil was successful in preventing only one of the observed diabetes-induced changes in atrial function, namely the supersensitivity of left atria to isoprenaline. Sorbinil treatment did, however, alter responses of control left and right atria in a manner similar to diabetes. In conclusion, streptozotocin-induced diabetes of 14 days duration was found to cause a number of alterations in the functioning of both left and right atria. ARI treatment with sorbinil failed to prevent all but one of these changes, and in addition altered responses of atria from control rats, having a similar effect to that produced by diabetes. These data suggest that sorbinil may have effects in addition to, and independent of, aldose reductase inhibition in the cardiovascular system.

In vitro responses of gallbladder muscle from patients with acalculous biliary pain.

BACKGROUND: Diagnosis and identification of patients with acalculous biliary pain, who would benefit from surgery, remains a significant clinical problem. The cholecystokinin (CCK) provocation test helps diagnosis, but lack of consistency limits its usefulness. AIM: To characterize the response of gallbladder muscle strips, from patients with acalculous biliary pain, to hormonal and muscarinic stimulation and to compare these with strips from gallstone patients and normal controls. PATIENTS: Eleven patients with acalculous biliary pain were studied, 5 had a positive CCK test. Eight gallbladders from gallstone patients and 6 from partial hepatectomies were used for comparison. METHODS: Muscle strips from the body and neck of the gallbladder were suspended in organ baths and dose-response curves were constructed for CCK-8 and carbachol. RESULTS: In the acalculous group the strips from the body were less sensitive to carbachol than those of the neck. CONCLUSION: Since we found no differences in the CCK responses for the groups, it casts doubt over the effectiveness of the CCK test to diagnose acalculous biliary pain. Since carbachol sensitivity was different, it might be that a similar test using muscarinic stimulation would help in the diagnosis of this difficult group of patients.

Urothelium-derived inhibitory factor(s) influences on detrusor muscle contractility in vitro.

The function of the bladder urothelium in modulating contractile responses of the underlying detrusor smooth muscle to muscarinic stimulation has been examined in the pig bladder. Saturation curves for [3H]-QNB binding demonstrated a greater muscarinic receptor density in the urothelium than in the detrusor smooth muscle. The presence of an intact urothelium on isolated bladder strips inhibited contractions induced by carbachol but not KCl. Contractions of a urothelium-denuded muscle strip were inhibited in the presence of a second bladder strip with an intact urothelium, but not if the second strip was denuded. The urothelium-induced inhibition of contractions was not prevented in the presence of L-NOARG, methylene blue, indomethacin, propranolol, suramin, TEA or apamin. The data suggest the presence of a diffusable, urothelium-derived inhibitory factor, which could not be identified but appears to be neither nitric oxide, a cyclo-oxygenase product, a catecholamine, adenosine, GABA nor an EDHF sensitive to apamin.

In vitro investigation of the bladder-vascular selectivity of levcromakalim and YM934 in human tissues.

OBJECTIVE: To examine the potencies of the potassium-channel openers (KCOs) levcromakalim and YM934 as relaxing agents on human detrusor and human mesenteric artery smooth muscle, and to determine their bladder-vascular selectivity in vitro. MATERIALS AND METHODS: Strips of human detrusor muscle and mesenteric artery (with the endothelium removed) were set up in physiological salt solution and the tension developed by the tissues recorded. Tissues were precontracted with a concentration of carbachol (detrusor) or phenylephrine (artery) which caused 80% of maximal contraction, and relaxation responses to levcromakalim and YM934 were then obtained. RESULTS: Both KCOs caused relaxation of the bladder detrusor muscle and the mesenteric artery. Maximal responses, when plotted as a percentage of the precontraction, were greater for both KCOs in the bladder muscle than the artery, but the differences were small and not statistically significant. The sensitivity (drug potency) of the detrusor muscle to the KCOs was more than twice that of the artery but this selectivity was only statistically significant for YM934. CONCLUSIONS: Only minor bladder-vascular selectivity for levcromakalim and YM934 could be detected in vitro. This suggests that neither drug would be tolerated clinically, although the results suggest that further development of bladder-selective KCO agents appears feasible.

In vitro contractility of stimulated and non-stimulated human gallbladder muscle.

In an attempt to define, more clearly, the nature of gallbladder contraction we obtained muscle strips from human gallbladder wall, removed at cholecystectomy. Samples were taken from various areas of the gallbladder to seek evidence of a dominant axis of contraction. The strips were stimulated with increasing concentrations of cholecystokinin-8 (CCK-8) and carbachol, and, to determine maximal contractile force, 0.25 M potassium chloride. No differences were seen between samples taken from the long-itudinal, circular and oblique axes. In a second series of experiments, samples were taken from the body and neck regions of the gallbladder. In these, it was seen that the samples from the body contracted more forcefully than those of the neck tissue and that they were more sensitive to carbachol stimulation. The difference in response to CCK-8 measured in the strips from the body and cystic duct/neck of the gallbladder cannot be explained by a difference in sensitivity to CCK-8, but is mainly due to the difference in the amount of muscle tissue present. Strips from the body are more sensitive to muscarinic stimulation that those from the neck. Overall, there is a functional difference in sensitivity between the body and neck which would serve to facilitate bile flow into the common bile duct during gallbladder contraction.

The influence of 17-beta-oestradiol and the natural oestrous cycle on alpha-adrenoceptor-mediated responses of the cardiovascular system in the rat.

The effect of 17 beta-oestradiol pretreatment and the natural oestrous cycle on responses of rat cardiac and vascular alpha-adrenoceptors was investigated. It was found that treatment with the oestrogen had no effect on cardiac adrenoceptor sensitivity, while pretreatment with the hormone caused a significant increase in vascular alpha 2-, but not vascular alpha 1-adrenoceptor responsiveness. Pressor responses of pithed rats to selective alpha 1- or alpha 2-adrenoceptor agonists did not alter, however, during the natural oestrous cycle. These findings suggest that changes in oestrogen levels are capable of altering alpha-adrenoceptor-mediated vascular responses but that the relatively small changes in oestrogen which may occur throughout the natural oestrous cycle do not appear to alter the responses of these vascular alpha-adrenoceptors.

Influence of adrenoceptor stimulation on aggregation of platelets from diabetic and control rats.

1. Studies of cardiac and vascular responses have previously demonstrated that diabetes influences the sensitivity of these tissues to adrenoceptor stimulation. Adrenoceptors are also present on platelets where they modulate aggregatory responses. The present study investigates the influence of diabetes on these platelet adrenoceptor-mediated responses. 2. Rats were made diabetic with streptozotocin and platelet aggregatory responses to ADP were examined 2 or 12 weeks later. Aggregation to ADP of platelets from 2-week-diabetic rats was similar to that of platelets from age-matched controls, but platelets from 12-week-diabetic animals exhibited an enhanced aggregation to ADP. 3. In all groups studied, beta-adrenoceptor stimulation with isoprenaline caused a concentration-dependent inhibition of aggregation to ADP, whilst alpha-adrenoceptor stimulation with adrenaline was found to potentiate aggregation to ADP. The degree of inhibition or potentiation was found to remain unchanged by diabetes of either 2 or 12 weeks duration. 4. Previously reported increases in cardiac beta-adrenoceptor sensitivity and aortic alpha-adrenoceptor sensitivity were confirmed 2-week-diabetic animals, but these sensitivity changes were not observed in 12-week-diabetic rats. 5. The results indicate that, unlike the heart and vasculature, the influences of adrenoceptor stimulation on platelet aggregation are not altered by diabetes, even when aggregation to ADP is enhanced.