Adrenergic relaxations in an in situ urinary bladder model evoked by stimulation of sensory pelvic and pudendal nerves in the rat.

Urinary bladder dysfunction might be related to disturbances at different levels of the micturition reflex arc. The current study aimed to further develop and evaluate a split bladder model for detecting and analysing relaxatory signalling in the rat urinary bladder. The model allows for discrimination between effects at the efferent and the afferent side of the innervation. In in vivo experiments, the stimulation at a low frequency (1 Hz) of the ipsilateral pelvic nerve tended to evoke relaxation of the split bladder half (contralateral side; -1.0 ± 0.4 mN; n = 5), in contrast to high frequency-evoked contractions. In preparations in which the contralateral pelvic nerve was cut the relaxation occurred at a wider range of frequencies (0.5-2 Hz). In separate experiments, responses to 1 and 2 Hz were studied before and after intravenous injections of propranolol (1 mg/kg IV). The presence of propranolol significantly shifted the relaxations into contractions. Also, electrical stimulation of the ipsilateral pudendal nerve evoked relaxations of similar magnitude as for the pelvic stimulations, which were also affected by propranolol. In control in vitro experiments, substances with ß-adrenoceptor agonism, in contrast to a selective α-agonist, evoked relaxations. The current study shows that the split bladder model can be used for in vivo studies of relaxations. In the model, reflex-evoked sympathetic responses caused relaxations at low intensity stimulation. The involvement of ß-adrenoceptors is supported by the sensitivity to propranolol and by the in vitro observations.

Soluble guanylate cyclase mediates the relaxation of healthy and inflamed bladder smooth muscle by aqueous nitric oxide.

Introduction: Due to its chemical properties, functional responses to nitric oxide (NO) are often difficult to examine. In the present study, we established a method to produce NO in an aqueous solution and validated its capacity to evoke functional responses in isolated rat bladders. Furthermore, we compared the NO responses to the commonly used NO donor sodium nitroprusside (SNP). We also investigated the impact of ongoing inflammation on the involvement of soluble guanylate cyclase (sGC) dependent signaling in NO relaxation. Methods: A setup to produce an aqueous NO solution was established, allowing the production of an aqueous solution containing a calculated NO concentration of 2 mM. Sixty male Sprague-Dawley rats received either no treatment (controls) or cyclophosphamide (CYP; 100 mg*kg-1 i.p., 60 h prior to the experiment) to induce experimental cystitis. Bladder strip preparations were mounted in organ baths and studied at basal tension or pre-contracted with methacholine (3 µM). Aqueous NO solution (40-400 µL; 2 mM corresponding to 4-40 µM) or SNP (1-1,000 µM) was added cumulatively in increasing concentrations. Relaxation to aqueous NO was also studied in the presence of the sGC inhibitor ODQ (0.25-25 µM). The expression of sGC was investigated by immunohistochemical analysis. Results: The NO solution caused functional relaxations in both controls and inflamed bladder preparations. NO-induced relaxations were significantly greater in inflamed bladder strips at basal tension, whereas no differences were seen in methacholine pre-contracted strips. In the presence of the sGC inhibitor ODQ in a high concentration, the NO-evoked relaxations were abolished in both control and inflamed preparations. At a lower concentration of ODQ, only NO relaxations in inflamed preparations were attenuated. Immunohistochemical analysis showed that sGC was expressed in the detrusor and mucosa, with a significantly lower expression in the inflamed detrusor. Conclusion: In the present study, we found that aqueous NO solution induces relaxation of the rat detrusor by activating soluble guanylate cyclase in both control and inflamed bladder strips. Induction of inflammation conceivably leads to decreased sGC expression in the detrusor, which may explain the different susceptibility towards inhibition of sGC in inflamed versus control tissue. The use of an aqueous NO solution should be further considered as a valuable complement to the pharmacological tools currently used.

Functional atropine sensitive purinergic responses in the healthy rat bladder.

While acetylcholine is regarded to be the main directly contractile transmitter substance in the urinary bladder, interactions with other transmitters likely occur. Presently, the interplay between purinergic and cholinergic signalling was investigated to unravel the involvement of the urothelium and efferent neurons in the functionally important purinergically evoked release of acetylcholine in vitro. Functional characterization of receptor subtypes involved in this interplay was also performed. In vitro organ bath experiments with electrical field stimulation (EFS) or administration of agonist were performed in the absence and presence of the neurotoxin tetrodotoxin (TTX; 5 × 10-7 M) and/or receptor antagonists, in intact and urothelium-denuded full thickness rat bladder strip preparations. Interestingly, functional contractions to ATP (10-6-10-3 M) remained unaffected by TTX, but were significantly lowered in the presence of the muscarinic antagonist atropine (10-6 M). However, in urothelium-denuded strip preparations, this latter phenomenon was not present and the ATP response remained unaltered. To rule out purinergic interference caused by break-down of ATP, experiments were performed in which the stable ATP-analogue αßMeATP (10-7-10-5 M) gave rise to functional atropine-sensitive contractions. Furthermore, contractions to ATP were not affected by P2Y6 purinoceptor blockade (by MRS2578; 10-7, 10-5 M), nor were relaxatory responses to ATP sensitive to atropine, PPADS (3 × 10-5 M) or αßMeATP. Lastly, relaxations to ADP (10-6-10-3 M) or NECA (10-8-10-5 M) were unaltered by the presence of atropine. To conclude, purinergic functional contractile, but not relaxatory, responses are supported by the cholinergic transmitter system in vitro, through non-neuronal mechanisms in the urothelium. Involved purinoceptors are of the P2X-subtype, most likely P2X1 and/or P2X3.

In vivo paracrine effects of ATP-induced urothelial acetylcholine in the rat urinary bladder.

Mechanical stretch of the urothelium induces the release of ATP that activates bladder afferent nerves. In the rat urinary bladder, ATP is also a contractile co-transmitter in the parasympathetic innervation. In isolated preparations, ATP evokes a urothelial release of acetylcholine that substantially contributes to ATP-evoked contractile responses. Currently we aimed to further examine the interactions of ATP and acetylcholine in the rat urinary bladder in two in vivo models. In the whole bladder preparation, atropine reduced ATP-evoked responses by about 50% in intact but denervated bladders, while atropine had no effect after denudation of the urothelium. In a split bladder preparation, reflex-evoked responses of the contralateral half were studied by applying stimuli (agonists or stretch) to the ipsilateral half. Topical administration of ATP and methacholine as well as of stretch induced contralateral reflex-evoked contractions. While topical administration of atropine ipsilaterally reduced the ATP- and stretch-induced contralateral contractions by 27 and 39%, respectively, the P2X purinoceptor antagonist PPADS reduced them by 74 and 84%. In contrary, the muscarinic M2-(M4)-selective receptor antagonist methoctramine increased the responses by 38% (ATP) and 75% (stretch). Pirenzepine (M1-selective antagonist) had no effect on the reflex. In vitro, in the absence of the reflex, methoctramine did not affect the ATP-induced responses. It is concluded that urothelial ATP potently induces the micturition reflex and stimulates urothelial release of acetylcholine. Acetylcholine subsequently acts on afferents and on the detrusor muscle. While muscarinic M2 and/or M4 receptors in the sensory innervation exert inhibitory modulation, muscarinic M3 receptors cause excitation.

Effects of caveolae depletion and urothelial denudation on purinergic and cholinergic signaling in healthy and cyclophosphamide-induced cystitis in the rat bladder.

Cholesterol rich membrane invaginations, caveolae, have important roles in various cellular activities, one of them being signal transduction. This signaling pathway seems to be affected during various bladder disorders and the current study aimed to elucidate the plausible involvement of caveolae mediated signal transduction during cyclophosphamide induced cystitis. Furthermore, the urothelial cholinergic part of ATP-evoked contractions and its possible link to caveolae were investigated. Cholinergic, as well as purinergic, contractile responses in rat urinary bladders were examined using a classic organ bath set-up with full-thickness strip preparations or a whole bladder model that enabled luminal administration of substances. Furthermore, sub groups with and without urothelium were examined. The expression of caveolin-1 was also tested using western blot and immunofluorescence. Caveolae cholesterol depletion by methyl-ß-cyclodextrin entailed a significant decrease of ATP-evoked bladder contractility. Interestingly, after muscarinic blockade the ATP induced contractions were significantly reduced in the same manner. Furthermore, this atropine-sensitive part of ATP-evoked responses was absent in denuded as well as inflamed bladders. A tendency towards a reduced expression of caveolin-1 was observed in rats with experimental cystitis. The cholinergic part of ATP-induced contractile responses seemed to be affected by urothelium denudation as well as caveolae depletion. Removing one of these structures nullifies the effect of the other, suggesting an important interaction between the urothelium and the caveolar structures. These effects are absent in inflamed animals and might be one pathophysiological aspect behind BPS/IC.

Urothelial acetylcholine involvement in ATP-induced contractile responses of the rat urinary bladder.

Both acetylcholine and adenosine 5'-triphosphate (ATP) are released from the urothelium. In in vivo experiments ATP has been shown to evoke contractile responses that are significantly reduced by atropine. Currently, we aimed to examine the cholinergic part of the ATP-evoked contractile response of normal and inflamed (cyclophosphamide-treated rats) bladders. A whole bladder preparation that enabled drug administration either outside or inside the urinary bladder was used. The responses were examined in bladders from control and cyclophosphamide-treated rats that were either intact or urothelium-denuded. The expression of choline acetyltransferase and carnitine acetyltransferase were examined by Western blotting of normal and inflamed bladders. Methacholine evoked larger contractions when administered to the outside of the bladder in comparison to instillation. For ATP, an opposite trend emerged. While atropine substantially reduced the ATP-induced responses at internal administration (7.4±1.1 and 3.7±0.9 mN at 10-3M; n=13; P<0.001), it had no effect when administered outside the bladder. The removal of the urothelium caused a similar reduction of the responses to internal administration of ATP as caused by atropine. In cyclophosphamide-treated rats, neither atropine nor urothelium-denudation had any effect on the ATP-evoked responses. No changes in the expressions of the acetylcholine synthesising enzymes were observed. The current study shows that ATP induces a release of urothelial acetylcholine that contributes to the purinergic contractile response in the rat urinary bladder. This atropine-sensitive part of the purinergic contractile response is absent in the inflamed bladder. This may be one pathological mechanism involved in bladder dysfunction.