Michael Winder

Signalling Molecules in the Urothelium

The urothelium was long considered to be a silent barrier protecting the body from the toxic effects of urine. However, today a number of dynamic abilities of the urothelium are well recognized, including its ability to act as a sensor of the intravesical environment. During recent years several pathways of these urothelial abilities have been proposed and a major part of these pathways includes release of signalling molecules. It is now evident that the urothelium represents only one part of the sensory web. Urinary bladder signalling is finely tuned machinery of signalling molecules, acting in autocrine and paracrine manner, and their receptors are specifically distributed among different types of cells in the urinary bladder. In the present review the current knowledge of the formation, release, and signalling effects of urothelial acetylcholine, ATP, adenosine, and nitric oxide in health and disease is discussed.

Cyclophosphamide‐induced alterations of the micturition reflex in a novel in situ urinary bladder model in the anesthetized rat

Cyclophosphamide‐induced cystitis alterations have been reported to occur both at efferent and afferent level in the micturition reflex arc. In particular, the stretching of the bladder wall causing urothelial release of ATP has been proposed as one of the pivotal mechanisms causing these alterations. To evaluate functional changes at efferent and afferent levels of the micturition reflex following cyclophosphamide treatment we have applied a novel in situ half bladder rat model.

Inhibition of Nitric Oxide Synthase Prevents Muscarinic and Purinergic Functional Changes and Development of Cyclophosphamide-Induced Cystitis in the Rat

Nitric oxide (NO) has pivotal roles in cyclophosphamide- (CYP-) induced cystitis during which mucosal nitric oxide synthase (NOS) and muscarinic M5 receptor expressions are upregulated. In cystitis, urothelial muscarinic NO-linked effects hamper contractility. Therefore we wondered if a blockade of this axis also affects the induction of cystitis in the rat. Rats were pretreated with saline, the muscarinic receptor antagonist 4-DAMP (1 mg/kg ip), or the NOS inhibitor L-NAME (30 mg/kg ip) for five days. 60 h before the experiments the rats were treated with saline or CYP. Methacholine-, ATP-, and adenosine-evoked responses were smaller in preparations from CYP-treated rats than from saline-treated ones. Pretreatment with 4-DAMP did not change this relation, while pretreatment with L-NAME normalized the responses in the CYP-treated animals. The functional results were strengthened by the morphological observations; 4-DAMP pretreatment did not affect the parameters studied, namely, expression of muscarinic M5 receptors, P1A1 purinoceptors, mast cell distribution, or bladder wall enlargement. However, pretreatment with L-NAME attenuated the differences. Thus, the current study provides new insights into the complex mechanisms behind CYP-induced cystitis. The NO effects coupled to urothelial muscarinic receptors have a minor role in the development of cystitis. Inhibition of NOS may prevent the progression of cystitis.

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

&NA; 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 10−3 M; 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.

In vivo studies of effects of antidepressants on parotid salivary secretion in the rat

Tricyclic antidepressants (TCA) are well-known xerogenic drugs, while antidepressants such as selective serotonin reuptake inhibitors (SSRI) are considered less xerogenic. The antimuscarinic effect of the TCAs has been considered to be the principal mechanism causing a dry mouth. Although the muscarinic receptor is commonly targeted by xerogenic pharmaceuticals, the salivation reflex arc may be affected at other levels as well. We currently wondered whether or not antidepressants exert an inhibition of the salivary reflex not only at the glandular level but at a central level as well. In this study, the effects of a TCA (clomipramine), a SSRI (citalopram) and a serotonin-noradrenaline reuptake inhibitor (SNRI; venlafaxine) were examined on reflex- (0.5M citric acid applied on the tongue) and methacholine-evoked salivary secretion. While all three compounds inhibited citric acid-evoked secretion (-40 to -60% at 5mg/kg i.v. of the antidepressants), only clomipramine inhibited methacholine-evoked secretion (-30% at 5mg/kg i.v.). On the contrary, both citalopram and venlafaxine increased the methacholine-evoked secretion (+44 to 49%). This was particularly obvious for the salivary protein output (>200%). In the presence of α- and β-adrenoceptor antagonists, the citalopram- and venlafaxine-induced increases were reduced. Thus, antidepressants irrespective of type may exert xerogenic effects by inhibiting the salivary reflex in the central nervous system. However, while TCAs may also hamper the secretory response by antimuscarinic effects, the SSRI and the SNRI groups of pharmaceuticals seem to lack this additional xerogenic mechanism indicating a better therapeutic profile and better opportunities for pharmacological treatment of drug-induced xerostomia.

A novel in situ urinary bladder model for studying afferent and efferent mechanisms in the micturition reflex in the rat

The search for new animal models to investigate both efferent and afferent levels of the micturition reflex, to better understand urinary dysfunctions, is of great importance. Therefore in this study we developed and characterized, by comparisons with a conventional whole bladder model, a novel in situ model.

Acetylcholinesterase inhibitors and drugs acting on muscarinic receptors - potential crosstalk of cholinergic mechanisms during pharmacological treatment.

Background Pharmaceuticals with targets in the cholinergic transmission have been used for decades and are still fundamental treatments in many diseases and conditions today. Both the transmission and the effects of the somatomotoric and the parasympathetic nervous systems may be targeted by such treatments. Irrespective of the knowledge that the effects of neuronal signalling in the nervous systems may include a number of different receptor subtypes of both the nicotinic and the muscarinic receptors, this complexity is generally overlooked when assessing the mechanisms of action of pharmaceuticals. Methods We have search of bibliographic databases for peer-reviewed research literature focused on the cholinergic system. Also, we have taken advantage of our expertise in this field to deduce the conclusions of this study. Results Presently, the life cycle of acetylcholine, muscarinic receptors and their effects are reviewed in the major organ systems of the body. Neuronal and non-neuronal sources of acetylcholine are elucidated. Examples of pharmaceuticals, in particular cholinesterase inhibitors, affecting these systems are discussed. The review focuses on salivary glands, the respiratory tract and the lower urinary tract, since the complexity of the interplay of different muscarinic receptor subtypes is of significance for physiological, pharmacological and toxicological effects in these organs. Conclusion Most pharmaceuticals targeting muscarinic receptors are employed at such large doses that no selectivity can be expected. However, some differences in the adverse effect profile of muscarinic antagonists may still be explained by the variation of expression of muscarinic receptor subtypes in different organs. However, a complex pattern of interactions between muscarinic receptor subtypes occurs and needs to be considered when searching for selective pharmaceuticals. In the development of new entities for the treatment of for instance pesticide intoxication, the muscarinic receptor selectivity needs to be considered. Reactivators generally have a muscarinic M2 receptor acting profile. Such a blockade may engrave the situation since it may enlarge the effect of the muscarinic M3 receptor effect. This may explain why respiratory arrest is the major cause for deaths by esterase blocking.

In vitro functional interactions of acetylcholine esterase inhibitors and muscarinic receptor antagonists in the urinary bladder of the rat

Obidoxime, a weak acetylcholine‐esterase (AChE) inhibitor, exerts muscarinic receptor antagonism with a significant muscarinic M2 receptor selective profile. The current examinations aimed to determine the functional significance of muscarinic M2 receptors in the state of AChE inhibition, elucidating muscarinic M2 and M3 receptor interaction. In the in vitro examinations, methacholine evoked concentration‐dependent bladder contractile and atrial frequency inhibitory responses. Although atropine abolished both, methoctramine (1 μmol/L) only affected the cholinergic response in the atrial preparations. However, in the presence of methoctramine, physostigmine, an AChE inhibitor, increased the basal tension of the bladder strip preparations (+68%), as well as the contractile responses to low concentrations of methacholine (< 5 μmol/L; +90–290%). In contrast to physostigmine, obidoxime alone raised the basal tension (+58%) and the responses to low concentrations of methacholine (< 5 μmol/L; +80–450%). Physostigmine concentration‐dependently increased methacholine‐evoked responses, similarly to obidoxime at low concentrations. However, at large concentrations (> 5 μmol/L), obidoxime, because of its unselective muscarinic receptor antagonism, inhibited the methacholine bladder responses. In conclusion, the current results show that muscarinic M2 receptors inhibit muscarinic M3 receptor‐evoked contractile responses to low concentrations of acetylcholine in the synaptic cleft. The muscarinic M2 and M3 receptor crosstalk could be a counteracting mechanism in the treatment of AChE inhibition when using reactivators, such as obidoxime.

Autonomic Receptor-Mediated Regulation of Production and Release of Nitric Oxide in Normal and Malignant Human Urothelial Cells

In the urinary bladder, the main source of NO seems to be the urothelium and the underlying suburothelium. In this study, we aimed to characterize how receptors in the human urothelium regulate the production and release of NO. For this, we cultured two human urothelial cell lines – the normal immortalized cell line UROtsa and the malignant cell line T24. These were treated with an array of agonists and antagonists with affinity for adrenergic, muscarinic and purinergic receptors. The production of NO and expression of nitric oxide synthase (NOS) was studied by immunocytochemistry and Western blotting. The amount of released NO was measured indirectly by detecting nitrite using amperometry and a Griess reaction kit. The results showed that NO, endothelial NOS and inducible NOS were predominantly produced and expressed in the close vicinity of the nucleus in untreated human urothelial cells. Upon treatment with a beta‐adrenoceptor agonist, but not any of the other agonists or antagonists, the pattern of NO production changed, showing a more even production throughout the cytosol. The pattern of expression of endothelial NOS changed in a similar way upon dobutamine treatment. The release of nitrite, as a measurement of NO, increased after treatment with dobutamine from 0.31 ± 0.029 to 1.97 ± 0.18 nmol and 0.80 ± 0.12 to 3.27 ± 0.24 nmol in UROtsa and T24, respectively. In conclusion, our results show that the expression of NOS and production of NO as well as the release of NO from human urothelial cells is regulated by beta‐adrenoceptor activation.

Local Change in Urinary Bladder Contractility Following CNS Dopamine Denervation in the 6-OHDA Rat Model of Parkinson’s Disease

Abstract Background: Urinary problems, including urinary frequency, urgency, and nocturia are some of the non-motor symptoms that correlate most with poor quality of life in Parkinson’s disease. However, the mechanism behind these symptoms is poorly understood, in particular regarding peripheral bladder pathophysiology following dopamine degeneration. Objective: In this study, we compared the contractile responsiveness of urinary bladder from the 6-OHDA unilateral rat model of Parkinson’s disease with that of normal untreated animals. Methods: The contractility of the urinary detrusor muscle was evaluated in bladder strip preparations using electrical field stimulation, and muscarinic and purinoceptor stimulations in an vitro organ bath setup. Results: Our data show that the overall contractile response following electrical field stimulation was significantly higher (43% at maximum contraction by 20–40 Hz stimulation) in the 6-OHDA-lesioned rats as compared to control animals. This increase was associated with a significant increase in the cholinergic contractile response, where the muscarinic agonist methacholine produced a 44% (at 10 −4 M concentration) higher response in the 6-OHDA-treated rats as compared to controls with a significant left-shift of the dose response. This indicates an altered sensitivity of the muscarinic receptor system following the specific central 6-OHDA-induced dopamine depletion. In addition a 36% larger contraction of strips from the 6-OHDA animals was also observed with purinoceptor activation using the agonist ATP (5×10 −3 M) during atropine treatment. Conclusions: Our data shows that it is not only the central dopamine control of the micturition reflex that is altered in Parkinson’s disease, but also the local contractile function of the urinary bladder. The current study draws attention to a mechanism of urinary dysfunction in Parkinson’s disease that has previously not been described.