New therapeutic targets to prevent benign prostatic enlargement and symptomatic progression to benign prostatic obstruction-ICI-RS 2023.

AIMS: Benign prostatic enlargement (BPE) can impact lower urinary tract function due to its potential progression to benign prostatic obstruction (BPO). Treatment options include removal of the obstruction by surgery or through use of therapeutics designed to slow growth or reduce tissue stress imposed by muscular stromal components. Inflammation and development of fibrosis can also raise intrinsic tissue stress within the gland, further impacting obstruction. Outflow tract obstruction can also impact emission and ejaculation if the obstruction persists. METHODS: This review summarizes an ICI-RS think tank considering novel drug treatments that might address BPO caused by progressive development of BPE, as well as manage decompensation changes to bladder function. RESULTS: Topics included recent advances in our understanding of pathological changes occurring to the prostate and other lower urinary tract tissues during progressive development of BPE, and how prevention or reversal might benefit from the identification of novel drug targets. These included contractile properties of prostatic tissues, the impact of BPE and its effects on bladder function, the deposition of intramural fibrotic tissue with protracted BPO, the role of inflammation in the development of BPE and its progression to BPO. In particular, we discussed current therapeutic options for treating BPE/BPO, and new therapeutic targets, what they treat and their advantage over current medications. CONCLUSION: Several new drug targets were identified, including soluble guanylate cyclase (sGC), the receptor for nitric oxide (NO•), and sGC activators that promotes sGC-mediated cGMP production when sGC is inactivated and unresponsive to NO•.

Nitric oxide signaling pathways in the normal and pathological bladder: Do they provide new pharmacological pathways?-ICI-RS 2023.

AIMS: The nitric oxide (NO•)/soluble guanylate cyclase/cyclic-GMP (cGMP) signaling pathway is ubiquitous and regulates several functions in physiological systems as diverse as the vascular, nervous, and renal systems. However, its roles in determining normal and abnormal lower urinary tract functions are unclear. The aim was to identify potential therapeutic targets associated with this pathway to manage lower urinary tract functional disorders. METHODS: This review summarizes a workshop held under the auspices of ICI-RS with a view to address these questions. RESULTS: Four areas were addressed: NO• signaling to regulate neurotransmitter release to detrusor smooth muscle; its potential dual roles in alleviating and exacerbating inflammatory pathways; its ability to act as an antifibrotic mediator; and the control by nitrergic nerves of lower urinary tract vascular dynamics and the contractile performance of muscular regions of the bladder wall. Central to much of the discussion was the role of the NO• receptor, soluble guanylate cyclase (sGC) in regulating the generation of the enzyme product, the second messenger cGMP. The redox state of sGC is crucial in determining its enzymic activity and the role of a class of novel agents, sGC activators, to optimize activity and to potentially alleviate the consequences of lower urinary tract disorders was highlighted. In addition, the consequences of a functional relationship between nitrergic and sympathetic nerves to regulate vascular dynamics was discussed. CONCLUSIONS: Several potential NO•-dependent drug targets in the lower urinary tract were identified that provide the basis for future research and translation to clinical trials.

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.

6-OHDA-Induced Changes in Colonic Segment Contractility in the Rat Model of Parkinson's Disease.

Background: Gastrointestinal dysfunction is one of the most common non-motor symptoms in Parkinson's disease (PD). The exact mechanisms behind these symptoms are not clearly understood. Studies in the well-established 6-hydroxydopamine (6-OHDA) lesioned rats of PD have shown altered contractility in isolated circular and longitudinal smooth muscle strips of distal colon. Contractile changes in proximal colon and distal ileum are nevertheless poorly studied. Moreover, segments may serve as better tissue preparations to understand the interplay between circular and longitudinal smooth muscle. This study aimed to compare changes in contractility between isolated full-thickness distal colon muscle strips and segments, and extend the investigation to proximal colon and distal ileum in the 6-OHDA rat model. Methods: Spontaneous contractions and contractions induced by electrical field stimulation (EFS) and by the non-selective muscarinic agonist methacholine were investigated in strip and/or segment preparations of smooth muscle tissue from distal and proximal colon and distal ileum in an in vitro organ bath comparing 6-OHDA-lesioned rats with Sham-operated animals. Key Results. Our data showed increased contractility evoked by EFS and methacholine in segments, but not in circular and longitudinal tissue strips of distal colon after central 6-OHDA-induced dopamine denervation. Changes in proximal colon segments were also displayed in high K+ Krebs-induced contractility and spontaneous contractions. Conclusions: This study further confirms changes in smooth muscle contractility in distal colon and to some extent in proximal colon, but not in distal ileum in the 6-OHDA rat model of PD. However, the changes depended on tissue preparation.

Teachers' Perspectives on Improving Online Seminars in Pharmacology: A Quantitative and Qualitative Study on Lessons Learned During the COVID-19 Pandemic.

The aim of this study is to evaluate teachers' perceptions of online seminars during COVID-19 pandemic to improve future courses in pharmacology. The study was performed as a questionnaire survey. A questionnaire that included 11 questions was used. A total of 14 online seminar teachers, of which 9 were senior teachers and 5 were PhD students, filled out the questionnaire. PhD students' and senior teachers' answers to questions 1-5 were compared statistically. The results of questions 6-10 were analysed qualitatively through thematic content analysis. There were no significant differences between senior teachers and PhD students in regard to the scores given to questions 1-5 in the questionnaire. Most (65%) teachers scored the online seminars lower than in person seminars. Interaction, communication, and group dynamics were mostly perceived to be less effective at online seminars compared to in person seminars. The main advantages of online seminars were time saving and flexibility. The main disadvantages of online seminars were reduced student interest, risk of monologue discussion and poorer communication without body language. Most teachers experienced minor technical problems with internet connection and sound quality. The teachers mentioned that better group dynamics, smaller groups, better chat functionality and clearer guidelines could help to improve online seminars. As an alternative to online seminars, blended-learning could be used. This way, one could appreciate both the richness of interactions in a face-to-face environment as well as the flexibility and convenience of online learning. Further studies comparing blended-learning and online teaching at seminars are needed to investigate this issue.

Treatment with the soluble guanylate cyclase activator BAY 60-2770 normalizes bladder function in an in vivo rat model of chronic prostatitis.

BACKGROUND AND PURPOSE: Chronic pelvic pain syndrome (CPPS) is a common and bothersome condition for which no pharmacological treatment options with acceptable efficacy exist. The aim of this study was to investigate the effects of the soluble guanylate cyclase (sGC) activator BAY 60-2770 and the COX-2 inhibitor celecoxib on bladder function in a rat model of CPPS. EXPERIMENTAL APPROACH: Forty-eight male Sprague-Dawley rats were intraprostatically injected with either saline, serving as control, or zymosan, to induce prostatitis. On days 8-20, the rats were treated with either dimethylsulphoxide (DMSO; vehicle), celecoxib, BAY 60-2770 or a combination of celecoxib and BAY 60-2770. Thereafter, micturition parameters were assessed in a metabolic cage and urine samples were collected. The following day, cystometry was performed. Subsequently, the urinary bladder and prostate were removed and examined histopathologically. KEY RESULTS: Induction of prostatitis led to a significant increase of micturition frequency and corresponding decrease of volume per micturition. These alterations were ameliorated by celecoxib, and completely normalized by BAY 60-2770. Induction of prostatitis led to a significantly increased number of non-voiding contractions, decreased bladder compliance and increased voiding time. These parameters were normalized by treatment with BAY 60-2770, either alone or in combination with celecoxib. The immunohistochemical analysis showed signs of prostate inflammation, but not bladder inflammation. CONCLUSION AND IMPLICATIONS: Induction of prostatitis led to significant impairment in bladder function. These alterations could be prevented by BAY 60-2770, alone or in combination with celecoxib. This is the first study to show that sGC activators could be a promising option for the treatment of CPPS.

Novel Insights Into Muscarinic and Purinergic Responses in Primary Cultures of Rat Lacrimal Gland Myoepithelial Cells.

Purpose: The functional characteristics of receptors that regulate lacrimal gland myoepithelial cells are still somewhat unclear. To date, mainly muscarinic receptors have been of interest; however, further knowledge is needed regarding their expression and functional roles. For this purpose, primary cultures of rat lacrimal gland myoepithelial cells were established and examined functionally. Methods: Rat lacrimal glands were excised, minced, and further digested, yielding mixtures of cells that were seeded in culturing flasks. After 4-6 weeks, primary monocultures of myoepithelial cells were established, verified by immunocytochemistry. The cells were stained for all muscarinic receptor subtypes (M1-M5) and examined functionally regarding intracellular [Ca2+] responses upon activation of muscarinic receptors. For methodological verification, purinergic functional responses were also studied. Results: Expression of muscarinic receptor subtypes M2-M5 was detected, whereas expression of muscarinic M1 receptors could not be shown. Activation of muscarinic receptors by the non-selective muscarinic agonist methacholine (3 × 10-11-10-3 M) did not cause a significant increase in intracellular [Ca2+]. However, activation of purinergic receptors by the non-selective purinergic agonist ATP (10-8-10-3 M) caused a concentration-dependent increase in intracellular [Ca2+] that could be blocked by the P2 antagonists PPADS and suramin. Conclusions: Primary cultures of rat lacrimal gland myoepithelial cells were established that displayed a heterogeneous expression of muscarinic receptors. Purinergic functional responses demonstrated a viable cell population. Upon treatment with methacholine, no significant increase in intracellular [Ca2+] could be detected, indicating that cholinergic activation of myoepithelial cells occurs via other intracellular messengers or is dependent on interaction with other cell types.

Cross-organ sensitization between the prostate and bladder in an experimental rat model of lipopolysaccharide (LPS)-induced chronic pelvic pain syndrome.

BACKGROUND: The aim of the current study was to investigate the effects of chronic prostatitis/chronic pelvic pain syndrome (CP/CPPS) on bladder function via prostate-to-bladder cross-sensitization in a rat model of lipopolysaccharide (LPS)-induced prostate inflammation. METHODS: Male rats were intraprostatically injected with LPS or saline, serving as control. Micturition parameters were examined in a metabolic cage 10 or 14 days later. Subsequently, to evaluate bladder function, cystometry was performed. Micturition cycles were induced by saline infusion and cholinergic and purinergic contractile responses were measured by intravenous injection with methacholine and ATP, respectively. Thereafter, the prostate and bladder were excised and assessed histopathologically for possible inflammatory changes. RESULTS: Metabolic cage experiments showed increased urinary frequency in rats with LPS-induced CP/CPPS. Cystometry showed a significant increase in the number of non-voiding contractions, longer voiding time and lower compliance in CP/CPPS animals compared to controls. Induction of CP/CPPS led to significantly reduced cholinergic and purinergic bladder contractile responses. Histopathological analysis demonstrated prostatic inflammation in CP/CPPS animals. There were no significant differences between the groups regarding the extent or the grade of bladder inflammation. Prostate weight was not significantly different between the groups. CONCLUSIONS: The present study shows that prostate-to-bladder cross-sensitization can be triggered by an infectious focus in the prostate, giving rise to bladder overactivity and alterations in both afferent and efferent signalling. Future studies are required to fully understand the underlying mechanisms.

Acute Inhibitory Effects of Antidepressants on Lacrimal Gland Secretion in the Anesthetized Rat.

Purpose: Patients that medicate with antidepressants commonly report dryness of eyes. The cause is often attributed to the anticholinergic properties of the drugs. However, regulation of tear production includes a substantial reflex-evoked component and is regulated via distinct centers in the brain. Further, the anticholinergic component varies greatly among antidepressants with different mechanisms of action. In the current study it was wondered if acute administration of antidepressants can disturb production of tears by affecting the afferent and/or central pathway. Methods: Tear production was examined in vivo in anesthetized rats in the presence or absence of the tricyclic antidepressant (TCA) clomipramine or the selective serotonin reuptake inhibitor (SSRI) escitalopram. The reflex-evoked production of tears was measured by challenging the surface of the eye with menthol (0.1 mM) and cholinergic regulation was examined by intravenous injection with the nonselective muscarinic agonist methacholine (1-5 µg/kg). Results: Acute administration of clomipramine significantly attenuated both reflex-evoked and methacholine-induced tear production. However, escitalopram only attenuated reflex-evoked tear production, while methacholine-induced production of tears remained unaffected. Conclusions: This study shows that antidepressants with different mechanisms of action can impair tear production by attenuating reflex-evoked signaling. Further, antimuscarinic actions are verified as a likely cause of lacrimal gland hyposecretion in regard to clomipramine but not escitalopram. Future studies on antidepressants with different selectivity profiles and mechanisms of action are required to further elucidate the mechanisms by which antidepressants affect tear production.

Prostate-to-bladder cross-sensitization in a model of zymosan-induced chronic pelvic pain syndrome in rats.

BACKGROUND: The aim of the present study was to investigate the effects of chronic prostatitis/chronic pelvic pain syndrome (CP/CPPS) on bladder function and pathophysiology. METHODS: To create a model for CPPS, rats were intraprostatically injected with zymosan or saline, serving as control. Metabolic cage experiments were performed 7, 14, or 21 days after zymosan injection and after 14 days in the control group. Thereafter, cystometry was performed in which simulated micturition cycles were induced by saline infusion and contractile responses to the cholinergic agonist methacholine and the purinergic agonist ATP were measured. Following cystometry, the prostate and urinary bladder were excised and assessed histopathologically for possible inflammatory changes. RESULTS: Metabolic cage data revealed a significantly increased urinary frequency in zymosan treated rats. Likewise, the volume per micturition was significantly lower in all CPPS groups compared to controls. Cystometry showed a significant increase in the number of nonvoiding contractions, longer voiding time, and a trend towards lower compliance in CPPS rats compared to controls. Induction of CPPS led to significantly reduced cholinergic and purinergic contractile responses. Histopathological analysis demonstrated prostatic inflammation in all CPPS groups, in particular in later stage groups. Both the extent and grade of bladder inflammation were significantly higher in CPPS groups compared to controls. CONCLUSIONS: The current findings demonstrate a potential prostate-to-bladder cross-sensitization leading to symptoms of bladder overactivity and signs of bladder inflammation. Future clinical studies are required to verify the outcomes of the current study and enable advancement of patient care.

Desipramine, commonly used as a noradrenergic neuroprotectant in 6-OHDA-lesions, leads to local functional changes in the urinary bladder and gastrointestinal tract in healthy rats.

The 6-hydroxydopamine (6-OHDA) rat model is one of the most common animal models of Parkinson's disease. When experimentally inducing dopaminergic neurodegeneration in the nigrostriatal pathway using 6-OHDA, the noradrenergic reuptake inhibitor desipramine is often systemically injected in order to protect against damages to the noradrenergic system in the brain. An increasing number of studies are focusing on understanding the pathophysiological changes underlying autonomic non-motor symptoms, in particular urinary bladder and gastrointestinal dysfunctions, of the disease. Several of these studies have investigated the contractile properties and the activation of smooth muscle in the 6-OHDA rat model. Since the injection of desipramine is commonly placed in close proximity to the urinary bladder and gastrointestinal tract, in the current study we wanted to understand if the drug alone has an effect. For this, we have injected a single dose (25 mg/kg) of desipramine either intraperitonially or subcutaneously and investigated smooth muscle contractility in vitro in the urinary bladder, proximal colon and distal ileum four weeks post injection. Our data show that desipramine significantly alters smooth muscle contractility of the urinary bladder and proximal colon in healthy rats. Conclusively, we suggest, based on our data, that desipramine should be omitted when using the 6-OHDA rat model to investigate smooth muscle function in Parkinson's disease research.

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.

Combination drug therapy against OAB normalizes micturition parameters and increases the release of nitric oxide during chemically induced cystitis.

Today, monotherapy is the most common pharmacological treatment option for patients suffering from overactive bladder (OAB). Recent reports have indicated potential benefits of combination therapy, using a muscarinic antagonist and a ß3 -adrenoceptor agonist. This may be of particular interest for therapy-resistant patients with OAB and concomitant cystitis. The objective of the current study was to assess how combination therapy affects bladder parameters in health and cystitis and if the efficacy of the drugs can be linked to altered release of nitric oxide (NO). Rats were pretreated with either a combination of the muscarinic antagonist tolterodine and ß3 -selective adrenoceptor agonist mirabegron or saline for 10 days. Forty-eight hours prior to assessing micturition parameters in a metabolic cage, the rats were intraperitoneally injected with cyclophosphamide, causing cystitis, or saline. Urine samples were collected and analyzed for NO content. Bladder contractile properties were assessed in an organ bath setup. Induction of cystitis led to bladder overactivity. Combination therapy normalized bladder parameters. Both induction of cystitis and drug treatment increased the release of NO. The innate contractile properties of the bladder were unaffected by combination therapy. This study demonstrates positive effects of combination drug therapy on symptoms of OAB, possibly indicating it to be a good option for treatment of OAB during concomitant cystitis. It remains to be determined if increased release of NO is crucial for successful pharmacological treatment of bladder overactivity during cystitis.

Student Characteristics Associated with Passing the Exam in Undergraduate Pharmacology Courses-a Cross-sectional Study in Six University Degree Programs.

Adequate knowledge in pharmacology is crucial in many professions but a non-negligible proportion of students fail the exams and knowledge of underlying factors is largely lacking. This study was performed to evaluate to what extent various factors are related to student performance in pharmacology-related courses in higher education, linking administrative data to attendance at non-mandatory teaching sessions and questionnaire replies. A total of 596 students (median age: 22 years; 70% female) were included from eight courses which are part of either the medical, pharmacy, dentistry, nursing, or biomedical analyst degree programs at the Sahlgrenska Academy, Gothenburg, Sweden. In all, 380 (64%) students passed the regular program- and course-specific exam. Multivariate logistic regression analysis revealed that a high participation rate in non-mandatory teaching sessions, as well as a perceived great interest in pharmacology, was associated with students' passing of the exam; adjusted odds ratio (95% confidence interval): 1.30 (1.19 to 1.42; per 10 percentage unit increase in attendance) and 3.38 (1.86 to 6.12), respectively. Working for wages during the course weeks and pre-university grades used in the program application were significant factors in subgroups of students, negatively and positively associated with the exam results, respectively. Age, having Swedish as a second language, and time spent studying were only associated with the exam result in the univariate analyses. To conclude, both students and teachers can contribute significantly to successful education within pharmacology, students by participating in the teaching sessions and teachers by encouraging students to find the subject interesting.

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.

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.

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 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 ß-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.