Patrik Aronsson

Assessment and Characterization of Purinergic Contractions and Relaxations in the Rat Urinary Bladder

The aim of the present study was to assess the purinoceptor functional responses of the urinary bladder by using isolated rat urinary bladder strip preparations. ATP elicited a transient bladder contraction followed by a sustained relaxation and ADP, UDP and UTP generated predominantly potent relaxations (relaxatory potencies: ADP = ATP > UDP = UTP). The ATP contractions were desensitized with the P2X(1/3) purinoceptor agonist/desensitizer alpha,beta-meATP and reduced by the P2 purinoceptor antagonist PPADS but unaffected by the P2 purinoceptor antagonist suramin. Electrical field stimulation (1-60 Hz) evoked frequency-dependent bladder contractions that were decreased by incubation with alpha,beta-meATP but not further decreased by PPADS. Suramin antagonized relaxations generated by UDP but not those by ADP, ATP or UTP. PPADS antagonized and tended to antagonize UTP and UDP relaxations, respectively, but did neither affect ADP nor ATP relaxations. ADP relaxations were insensitive to the P2Y(1) purinoceptor antagonist MRS 2179 and the ATP-sensitive potassium channel antagonist glibenclamide. The ATP relaxations were inhibited by the P1 purinoceptor antagonist 8-p-sulfophenyltheophylline but unaffected by the A2A adenosine receptor antagonist 8-(3-chlorostyryl)caffeine and glibenclamide. Adenosine evoked relaxations that were antagonized by the A2B adenosine receptor antagonist PSB 1115. Thus, in the rat urinary bladder purinergic contractions are elicited predominantly by stimulation of the P2X(1) purinoceptors, while UDP/UTP-sensitive P2Y purinoceptor(s) and P1 purinoceptors of the A2B adenosine receptor subtype are involved in bladder relaxation.

Muscarinic receptor subtypes involved in urothelium-derived relaxatory effects in the inflamed rat urinary bladder

Functional studies have shown altered cholinergic mechanisms in the inflamed bladder, which partly depend on muscarinic receptor-induced release of nitric oxide (NO). The current study aimed to characterize which muscarinic receptor subtypes that are involved in the regulation of the nitrergic effects in the bladder cholinergic response during cystitis. For this purpose, in vitro examinations of carbachol-evoked contractions of inflamed and normal bladder preparations were performed. The effects of antagonists with different selectivity for the receptor subtypes were assessed on intact and urothelium-denuded bladder preparations. In preparations from cyclophosphamide (CYP; in order to induce cystitis) pre-treated rats, the response to carbachol was about 75% of that of normal preparations. Removal of the urothelium or administration of a nitric oxide synthase inhibitor re-established the responses in the inflamed preparations. Administration of 4-diphenylacetoxy-N-methylpiperidine (4-DAMP) inhibited the carbachol-induced contractile responses of preparations from CYP pre-treated rats less potently than controls. Pirenzepine and p-fluoro-hexahydro-sila-diphenidol (pFHHSiD) affected the carbachol-induced contractile responses to similar extents in preparations of CYP pre-treated and control rats. However, the Schild slopes for the three antagonists were all significantly different from unity in the preparations from CYP pre-treated rats. Again, L-NNA or removal of the urothelium eliminated any difference compared to normal preparations. This study confirms that muscarinic receptor stimulation in the inflamed rat urinary bladder induces urothelial release of NO, which counteracts detrusor contraction.

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.

Changes in muscarinic receptors in the toad urothelial cell line TBM-54 following acrolein treatment.

1 In cyclophosphamide‐induced cystitis in the rat, cholinergic function of the bladder and muscarinic receptor expression are altered. In the present study, we investigated whether the toad urothelial cell line TBM‐54 expresses functional muscarinic receptors and whether changes in muscarinic receptors can be induced in vitro by treating cells with acrolein, a metabolite of cyclophosphamide causing cystitis. 2 The occurrence of muscarinic receptors on cells was assessed by microphysiometry, a method analysing receptor function by measuring changes in the extracellular acidity rate (ECAR) in response to receptor stimulation. 3 Challenging untreated cells with the muscarinic receptor agonist carbachol gave rise to a concentration‐dependent increase in changes in ECAR, with a maximal response at 1 mmol/L carbachol of 51 ± 6%. Pre‐incubating cells with different muscarinic receptor antagonists (i.e. pirenzepine (M1 receptor selective), methoctramine (M2/M4 receptor selective) and 4‐diphenylacetoxy‐N‐methylpiperidine methobromide (4‐DAMP; M3/M1/M5 receptor selective)), gave rise to a concentration‐dependent decrease in the effects of carbachol (0.5 mmol/L) on changes in ECAR. 4 Western blot analysis was used to determine the expression of all muscarinic receptor subtypes (M1–M5) by the cell line. Following acrolein treatment, cells were markedly less sensitive to carbachol and the expression of muscarinic M2 receptors was decreased, whereas the expression of muscarinic M3 receptors was increased. 5 In conclusion, the urothelial cell line TBM‐54 expresses functional muscarinic receptors and exposure to acrolein leads to a modulation in the expression of muscarinic receptors. Consequently, acrolein may have direct effects on muscarinic receptor function and expression that contribute to the pathogenesis of cyclophosphamide‐induced cystitis.

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.

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.

On the pedagogy of pharmacological communication: a study of final semester health science students.

BackgroundThere is a need to improve design in educational programmes for the health sciences in general and in pharmacology specifically. The objective of this study was to investigate and problematize pharmacological communication in educational programmes for the health sciences.MethodsAn interview study was carried out where final semester students from programmes for the medical, nursing and specialist nursing in primary health care professions were asked to discuss the pharmacological aspects of two written case descriptions of the kind they would meet in their everyday work. The study focused on the communication they envisaged taking place on the concerns the patients were voicing, in terms of two features: how communication would take place and what would be the content of the communication. A phenomenographic research approach was used.ResultsThe results are presented as outcome spaces, sets of categories that describe the variation of ways in which the students voiced their understanding of communication in the two case descriptions and showed the qualitatively distinct ways in which the features of communication were experienced.ConclusionsThe results offer a base of understanding the students’ perspectives on communication that they will take with them into their professional lives. We indicate that there is room for strengthening communication skills in the field of pharmacology, integrating them into programmes of education, by more widely implementing a problem-based, a case-oriented or role-playing pedagogy where final year students work across specialisations and there is a deliberate effort to evoke and assess advanced conceptions and skills.

The understanding of core pharmacological concepts among health care students in their final semester.

BackgroundThe overall aim of the study was to explore health care students´ understanding of core concepts in pharmacology.MethodAn interview study was conducted among twelve students in their final semester of the medical program (n = 4), the nursing program (n = 4), and the specialist nursing program in primary health care (n = 4) from two Swedish universities. The participants were individually presented with two pharmacological clinically relevant written patient cases, which they were to analyze and propose a solution to. Participants were allowed to use the Swedish national drug formulary. Immediately thereafter the students were interviewed about their assessments. The interviews were audio-recorded and transcribed verbatim. A thematic analysis was used to identify units of meaning in each interview. The units were organized into three clusters: pharmacodynamics, pharmacokinetics, and drug interactions. Subsequent procedure consisted of scoring the quality of students´ understanding of core concepts. Non-parametric statistics were employed.ResultsThe study participants were in general able to define pharmacological concepts, but showed less ability to discuss the meaning of the concepts in depth and to implement these in a clinical context. The participants found it easier to grasp concepts related to pharmacodynamics than pharmacokinetics and drug interactions.ConclusionThese results indicate that education aiming to prepare future health care professionals for understanding of more complex pharmacological reasoning and decision-making needs to be more focused and effective.

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.

ATP evokes cholinergic contraction in the healthy, but not inflamed, intact rat urinary bladder

Background: Over the past years increasing evidence suggesting an altered role of purinergic signaling during pathological conditions has been presented. Furthermore, there have been speculations regarding a possible link between purinergic and cholinergic functional mechanisms. In order to further investigate this, an “intact (whole) bladder” in vitro-setup has presently been employed. Aim: To elucidate the role of the urothelium and the functional link between purinergic and cholinergic signaling in the healthy and inflamed intact rat urinary bladder. Methods: Intact urinary bladders were excised from anesthetized rats; controls or cyclophosphamide-treated (100 mg/kg 60 h before the experiment to induce cystitis). Two catheters were inserted into the bladder for administration of substances and outflow, respectively. Collagenase I was used to denude the urothelium. Functional studies were conducted in an organ bath system. Agonists and antagonists could be administered both into the surrounding Krebs solution and intravesically (via the catheters). Results and Conclusions: When instilling ATP into the intact bladder, a contraction was observed (5.8 ± 0.9 mN; 1 mM; n = 19) which, interestingly, was significantly decreased in the presence of atropine (1 μM; 2.2 ± 0.6 mN, p b 0.01; n = 11). This difference was not observed neither in inflamed (2.8 ± 0.4 and 2.4 ± 0.1mN; n = 3-8), nor in urotheliumdenuded bladders (3.0 ± 1.3 and 3.3 ±2.0 mN; n = 4-8). This preliminary study shows the delicate interactions between the two transmitter systems, namely that ATP partly induces detrusor muscle contractions indirectly by stimulating the release of acetylcholine. Also, the urothelium seems to be involved in this cholinergic response, which appears to be diminished during inflammation.