Embracing the Versatility of Botulinum Neurotoxins in Conventional and New Therapeutic Applications.

Botulinum neurotoxins (BoNTs) have been used for almost half a century in the treatment of excessive muscle contractility. BoNTs are routinely used to treat movement disorders such as cervical dystonia, spastic conditions, blepharospasm, and hyperhidrosis, as well as for cosmetic purposes. In addition to the conventional indications, the use of BoNTs to reduce pain has gained increased recognition, giving rise to an increasing number of indications in disorders associated with chronic pain. Furthermore, BoNT-derived formulations are benefiting a much wider range of patients suffering from overactive bladder, erectile dysfunction, arthropathy, neuropathic pain, and cancer. BoNTs are categorised into seven toxinotypes, two of which are in clinical use, and each toxinotype is divided into multiple subtypes. With the development of bioinformatic tools, new BoNT-like toxins have been identified in non-Clostridial organisms. In addition to the expanding indications of existing formulations, the rich variety of toxinotypes or subtypes in the wild-type BoNTs associated with new BoNT-like toxins expand the BoNT superfamily, forming the basis on which to develop new BoNT-based therapeutics as well as research tools. An overview of the diversity of the BoNT family along with their conventional therapeutic uses is presented in this review followed by the engineering and formulation opportunities opening avenues in therapy.

Cell Plasticity in a Mouse Model of Benign Prostate Hyperplasia Drives Amplification of Androgen-Independent Epithelial Cell Populations Sensitive to Antioxidant Therapy.

Benign prostate hyperplasia (BPH) is caused by the nonmalignant enlargement of the transition zone of the prostate gland, leading to lower urinary tract symptoms. Although current medical treatments are unsatisfactory in many patients, the limited understanding of the mechanisms driving disease progression prevents the development of alternative therapeutic strategies. The probasin-prolactin (Pb-PRL) transgenic mouse recapitulates many histopathological features of human BPH. Herein, these alterations parallel urodynamic disturbance reminiscent of lower urinary tract symptoms. Single-cell RNA-sequencing analysis of Pb-PRL mouse prostates revealed that their epithelium mainly includes low-androgen signaling cell populations analogous to Club/Hillock cells enriched in the aged human prostate. These intermediate cells are predicted to result from the reprogramming of androgen-dependent luminal cells. Pb-PRL mouse prostates exhibited increased vulnerability to oxidative stress due to reduction of antioxidant enzyme expression. One-month treatment of Pb-PRL mice with anethole trithione (ATT), a specific inhibitor of mitochondrial ROS production, reduced prostate weight and voiding frequency. In human BPH-1 epithelial cells, ATT decreased mitochondrial metabolism, cell proliferation, and stemness features. ATT prevented the growth of organoids generated by sorted Pb-PRL basal and LSCmed cells, the two major BPH-associated, androgen-independent epithelial cell compartments. Taken together, these results support cell plasticity as a driver of BPH progression and therapeutic resistance to androgen signaling inhibition, and identify antioxidant therapy as a promising treatment of BPH.

A snake toxin as a theranostic agent for the type 2 vasopressin receptor.

Rationale: MQ1, a snake toxin which targets with high nanomolar affinity and absolute selectivity for the type 2 vasopressin receptor (V2R), is a drug candidate for renal diseases and a molecular probe for imaging cells or organs expressing V2R. Methods: MQ1's pharmacological properties were characterized and applied to a rat model of hyponatremia. Its PK/PD parameters were determined as well as its therapeutic index. Fluorescently and radioactively labeled MQ1 were chemically synthesized and associated with moderate loss of affinity. MQ1's dynamic biodistribution was monitored by positron emission tomography. Confocal imaging was used to observe the labeling of three cancer cell lines. Results: The inverse agonist property of MQ1 very efficiently prevented dDAVP-induced hyponatremia in rats with low nanomolar/kg doses and with a very large therapeutic index. PK (plasma MQ1 concentrations) and PD (diuresis) exhibited a parallel biphasic decrease. The dynamic biodistribution showed that MQ1 targets the kidneys and then exhibits a blood and kidney biphasic decrease. Whatever the approach used, we found a T1/2α between 0.9 and 3.8 h and a T1/2ß between 25 and 46 h and demonstrated that the kidneys were able to retain MQ1. Finally, the presence of functional V2R expressed at the membrane of cancer cells was, for the first time, demonstrated with a specific fluorescent ligand. Conclusion: As the most selective V2 binder, MQ1 is a new promising drug for aquaresis-related diseases and a molecular probe to visualize in vitro and in vivo V2R expressed physiologically or under pathological conditions.

Combined Sabal and Urtica Extracts (WS® 1541) Exert Anti-proliferative and Anti-inflammatory Effects in a Mouse Model of Benign Prostate Hyperplasia.

WS® 1541 is a phytopharmaceutical drug combination containing a lipophilic extract from fruits of Sabal serrulata (WS® 1473) and an aqueous ethanolic extract from roots of Urtica dioica (WS® 1031). It is approved in several countries worldwide for the treatment of lower urinary tract syndrome (LUTS) linked to benign prostate hyperplasia (BPH). Clinical studies have demonstrated the efficacy of this unique combination in the treatment of BPH-related LUTS. However, its mechanisms of action in vivo remain partly uncharacterized. The aim of this study was to take advantage of a validated mouse model of BPH to better characterize its growth-inhibitory and anti-inflammatory properties. We used the probasin-prolactin (Pb-PRL) transgenic mouse model in which prostate-specific overexpression of PRL results in several features of the human disease including tissue hypertrophy, epithelial hyperplasia, increased stromal cellularity, inflammation, and LUTS. Six-month-old heterozygous Pb-PRL male mice were randomly distributed to five groups (11-12 animals/group) orally treated for 28 consecutive days with WS® 1541 (300, 600, or 900 mg/kg/day), the 5α-reductase inhibitor finasteride used as reference (5 mg/kg/day) or vehicle (olive oil 5 ml/kg/day). Administration of WS® 1541 was well tolerated and caused a dose-dependent reduction of prostate weight (vs. vehicle) that was statistically significant at the two highest doses. This effect was accompanied by a reduction in prostate cell proliferation as assessed by lower Ki-67 expression (qPCR and immunohistochemistry). In contrast, finasteride had no or only a mild effect on these parameters. The growth-inhibitory activity of WS® 1541 was accompanied by a strong anti-inflammatory effect as evidenced by the reduced infiltration of cells expressing the leukocyte common antigen CD45. In sharp contrast, finasteride significantly increased the prostate inflammatory status according to this readout. Molecular profiling (qPCR) of 23 selected pro-inflammatory genes confirmed the strong anti-inflammatory potency of WS® 1541 compared to finasteride. Since treatment of WS® 1541 did not interfere with transgene expression and activity in the prostate of Pb-PRL mice, the effects observed in this study are entirely attributable to the intrinsic pharmacological action of the drug combination.

Netupitant, a Potent and Highly Selective NK1 Receptor Antagonist, Alleviates Acetic Acid-Induced Bladder Overactivity in Anesthetized Guinea-Pigs.

Introduction. Tachykinins potently contract the isolated urinary bladder from a number of animal species and play an important role in the regulation of the micturition reflex. On the guinea-pig isolated urinary bladder we examined the effects of a new potent and selective NK1 receptor antagonist (netupitant) on the contractions induced by a selective NK1 receptor agonist, SP-methylester (SP-OMe). Moreover, the effects of netupitant and another selective NK1 antagonist (L-733,060) were studied in anesthetized guinea-pigs using two experimental models, the isovolumetric bladder contractions and a model of bladder overactivity induced by intravesical administration of acetic acid (AA). Methods and Results. Detrusor muscle strips were mounted in 5 mL organ baths and isometric contractions to cumulative concentrations of SP-OME were recorded before and after incubation with increasing concentrations of netupitant. In anesthetized female guinea-pigs, reflex bladder activity was examined under isovolumetric conditions with the bladder distended with saline or during cystometry using intravesical infusion of AA. After a 30 min stabilization period, netupitant (0.1-3 mg/kg, i.v.) or L-733,060 (3-10 mg/kg, i.v.) were administered. In the detrusor muscle, netupitant produced a concentration-dependent inhibition (mean pKB = 9.24) of the responses to SP-OMe. Under isovolumetric conditions, netupitant or L-733,060 reduced bladder contraction frequency in a dose-dependent manner, but neither drug changed bladder contraction amplitude. In the AA model, netupitant dose-dependently increased intercontraction interval (ICI) but had no effect on the amplitude of micturition (AM). L-733,060 dose-dependently increased ICI also but this effect was paralleled by a significant reduction of AM. Conclusion. Netupitant decreases the frequency of reflex bladder contractions without altering their amplitude, suggesting that this drug targets the afferent limb of the micturition reflex circuit and therefore may be useful clinically in treating bladder overactivity symptoms.

The actions of prolonged exposure to cholinergic agonists on isolated bladder strips from the rat.

The present study was done to explore the cholinergic systems operating in the wall of the isolated rat bladder. In a first set of experiments, bladder strips in vitro were subjected to cumulative concentration-response curve (CRC) to non-selective muscarine agonist carbachol or the partially M2>M3 selective agonist arecaidine to establish optimal concentration to be used thereafter. In a second set of experiments, the effects of drugs (solifenacin, isoproterenol, and mirabegron) were tested on urinary bladder contraction induced by the non-selective muscarinergic agonist carbachol. For both agonists, the contractile responses are qualitatively similar: an initial transient rise in tension followed by complex bursts of high-frequency small 'micro'-contractions superposed on a tonic contraction, with immediate transient 'rebound' contraction after the agonist is washed from the preparation. This rebound contraction is greater with carbachol than arecaidine. Components of the responses to cholinergic stimulation, notably the micro-contractions, were found to be differently stimulated and inhibited by the M3>M2 selective antagonist solifenacin and by the ß-adrenoceptor agonists isoprenaline and mirabegron. A physiological role for the muscarinic dependent phasic contractions and the micro-anatomical elements that might be involved are not known but may be related to non-voiding activity observed during filling cystometry in conscious animals related to afferent discharge and possibly sensation. Furthermore, suggestions for the potential impact of these findings and design of further studies in relation to bladder physiology, pharmacology, and pathology are discussed.

The effect of 5-HT and electrical field stimulation on the contractility of the whole isolated urinary bladder of Suncus murinus.

The present study used the whole isolated urinary bladder of Suncus murinus, to investigate the effect of exogenously added serotonin (5-HT) and electrical field stimulation (EFS) in the absence and presence of methysergide, a 5-HT1/2/7 receptor antagonist or the selective 5-HT7 receptor antagonist, SB269970. Further experiments investigated the involvement of potassium channel, cholinergic and purinergic systems in mediating the contractile response to EFS. Pre-treatment with methysergide reduced and increased the contractile responses to 5-HT and EFS, respectively. Pre-treatment with SB269970 increased the responses to 5-HT without modifying the EFS-induced contractions. EFS-induced contractions were not modified by pre-treatment with atropine (10µM), α-ß-methylene ATP or glibenclamide. EFS-induced contractions were attenuated by cromakalim (10µM) or atropine (0.1 µM). In conclusion, the 5-HT2 receptors are likely to play a role in mediating the contractile response to 5-HT in detrusor muscle. Furthermore, EFS-induced contractions are mediated through cholinergic and an unknown neurotransmitter which is modulated by K(ATP) channels in the detrusor muscle of Suncus murinus.

Modulation of nerve-evoked contractions by ß3-adrenoceptor agonism in human and rat isolated urinary bladder.

Activation of ß3-adrenoceptors has been shown to have a direct relaxant effect on urinary bladder smooth muscle from both rats and humans, however there are very few studies investigating the effects of ß3-adrenoceptor agonists on nerve-evoked bladder contractions. Therefore in the current study, the role of ß3-adrenoceptors in modulating efferent neurotransmission was evaluated. The effects of ß3-adrenoceptor agonism on neurogenic contractions induced by electrical field stimulation (EFS) were compared with effects on contractions induced by exogenous acetylcholine (Ach) and αß-methylene adenosine triphosphate (αß-meATP) in order to determine the site of action. Isoproterenol inhibited EFS-induced neurogenic contractions of human bladder (pD2=6.79; Emax=65%). The effect of isoproterenol was selectively inhibited by the ß3-adrenoceptor antagonist L-748,337 (pKB=7.34). Contractions induced by exogenous Ach (0.5-1µM) were inhibited 25% by isoproterenol (3µM) while contractions to 10Hz in the same strip were inhibited 67%. The selective ß3-adrenoceptor agonist CL-316,243 inhibited EFS-induced neurogenic contractions of rat bladder (pD2=7.83; Emax=65%). The effects of CL-316,243 were inhibited in a concentration dependent manner by L-748,337 (pA2=6.42). Contractions induced by exogenous Ach and αß-meATP were significantly inhibited by CL-316,243, 29% and 40%, respectively. These results demonstrate that the activation of ß3-adrenoceptors inhibits neurogenic contractions of both rat and human urinary bladder. Contractions induced by exogenously applied parasympathetic neurotransmitters are also inhibited by ß3-agonism however the effect is clearly less than on neurogenic contractions (particularly in human), suggesting that in addition to a direct effect on smooth muscle, activation of prejunctional ß3-adrenoceptors may inhibit neurotransmitter release.

Relevance of the cyclophosphamide-induced cystitis model for pharmacological studies targeting inflammation and pain of the bladder.

This work aimed at establishing the relevance of using the in vivo model of cyclophosphamide (CYP)-induced bladder inflammation in rats for in vivo pharmacological studies. Specifically, we measured visceral nociception, identified key inflammatory mediators and evaluated the effects of relevant pharmacological treatments. Cystitis was induced in female rats by a single CYP injection. Sensitivity of the lower abdomen to von Frey mechanical stimulation was determined as a nociceptive parameter. Bladders were assessed for weight, wall thickness and macroscopic damage. Inflammatory mediators were quantified in bladders and urines. The effects of aspirin, ibuprofen and morphine were investigated on all these parameters. A single CYP injection increased nociceptive scores and decreased nociceptive threshold in response to mechanical stimuli between 1 and 4h post-administration. Increased bladder weight and wall thickness were associated with edema and hemorrhage. Bladder levels of IL-1ß, IL-6, MCP-1 and VCAM, and urinary levels of PGE2 were increased. In contrast, a decrease in the urinary metabolites, indoxyl sulfate and pantothenic acid, was observed. Aspirin, ibuprofen and morphine decreased CYP-induced referred visceral pain. Aspirin and ibuprofen also reversed the increased wall thickness, macroscopic damage and levels of IL-1ß, IL-6 and PGE2, and the decreased panthotenic acid levels. In contrast, morphine increased wall thickness, edema, hemorrhage, and bladder IL-6 and MCP-1 levels. This work presents a new and reliable method to evaluate visceral sensitivity in rats, and new relevant biomarkers identified in the bladder and urine to measure inflammation and pain parameters for in vivo pharmacological studies.

Fenoterol functionally activates the ß3-adrenoceptor in human urinary bladder, comparison with rat and mouse: implications for drug discovery.

Fenoterol has been reported to be a potent and selective ß(2)-adrenoceptor agonist and is currently used clinically to treat asthma. Electrical field stimulation (EFS) of isolated urinary bladder mimics the voiding contraction by stimulating parasympathetic nerves, resulting in neurogenic contractions. To determine if stimulation of ß(2)-adrenoceptors can inhibit this response, fenoterol was tested against EFS-induced contractions in human isolated urinary bladder and compared with mouse and rat. Bladder strips were mounted in organ baths and reproducible contractions induced by EFS. Fenoterol was added cumulatively in the presence of the ß(2)-adrenoceptor antagonist ICI118551 or the ß(3)-adrenoceptor antagonist L-748337. Fenoterol inhibited neurogenic contractions in all three species in a concentration-dependent manner with pEC(50) values of 6.66 ± 0.11, 6.86 ± 0.06 and 5.71 ± 0.1 in human, mouse and rat respectively. In human bladder strips ICI118551 (100 nM) did not affect responses to fenoterol, while L-748337 (0.3-3 µM) produced rightward shifts of the concentration-response curves with a pA(2) value of 8.10. In mouse bladder strips ICI118551 (30 nM) blocked the inhibitory effect of fenoterol (pA(2)=8.80), while L-748337 (10 µM) inhibited the response with a pA(2) of 5.79. In rat bladder ICI118551 (30 nM) was without effect, while L-748,337 (10 µM) inhibited the response to fenoterol with a pA(2) of 5.40. From these results it is clear that fenoterol potently activates ß(3)-adrenoceptors in human isolated urinary bladder to inhibit EFS-induced contractions. Fenoterol also activates ß(3)-adrenoceptors in rat, but ß(2)-adrenoceptors in mouse bladder to inhibit EFS-induced contractions.

Modulation of non-voiding activity by the muscarinergic antagonist tolterodine and the ß(3)-adrenoceptor agonist mirabegron in conscious rats with partial outflow obstruction.

UNLABELLED: Experimental urethral obstruction in rats alters micturition patterns with non-voiding activity (NVA) during filling cystometry, showing similarity to that observed in human detrusor overactivity. Several drug classes with therapeutic potential in overactive bladder in humans have been tested in this model in rats, rabbits or guinea pigs, but no detailed analysis of drug effects on cystometric patterns has been published. The present study uses a rat model of overactivity with partial bladder outflow obstruction (BOO) in combination with the procedures to analyse NVA to study the effects of the anticholinergic drug tolterodine and the novel ß(3)-adrenoceptor agonist mirabegron. The current data for the first time show that NVA in rats with BOO is sensitive to both the muscarinergic antagonist tolterodine and the ß(3)-adrenoceptor agonist mirabegron, but with clear differences between the two drugs: during progression of bladder filling, tolterodine affected both the amplitude and frequency of NVA whereas mirabegron affected primarily the frequency. In addition, tolterodine dose-dependently reduced voiding contractions, while mirabegron did not. A model is proposed to account for these observations where both agents act on a 'pacemaker-like' mechanism which is sensitive to cholinergic excitatory and beta-adrenergic inhibitory inputs. Such concepts could provide insights into the nature of overactive bladder and the site of action of key therapeutic drugs. OBJECTIVE: To investigate the hypothesis that tolterodine and the ß(3)-adrenoceptor agonist mirabegron exert their actions on the motor component of the motor/sensory system in the bladder wall: non-voiding activity (NVA). MATERIALS AND METHODS: The present study used standard cystometric techniques and a conscious rat model of partial bladder outflow obstruction (BOO). A single dose of either tolterodine (0.01, 0.1 0.3 or 1.0 mg/kg) or mirabegron (0.03, 0.1, 0.3, 1.0 or 3.0 mg/kg) was given i.v. to each animal. RESULTS: In the dose ranges used, tolterodine reduced the voiding contraction amplitude, whereas mirabegron did not. Non-voiding activity consisted of small (<0.6 mmHg) and large (>0.6 mmHg) transients. As a fill progressed, both tolterodine and mirabegron reduced the cumulative activity of the large non-voiding contractions, but had little effect on the small transients. Tolterodine affected both the amplitude and frequency of NVA, whereas mirabegron affected primarily the frequency. CONCLUSIONS: Non-voiding activity is sensitive to muscarinergic antagonists and ß(3)-adrenoceptor agonists, but there are clear differences between the two drugs. A model is proposed to account for these observations where both agents act on a 'pacemaker-like' mechanism with cholinergic excitatory and adrenergic inhibitory inputs. Such concepts may provide insights into the nature of overactive bladder and the site of action of key therapeutic drugs.

G protein-coupled receptors, an unexploited animal toxin targets: Exploration of green mamba venom for novel drug candidates active against adrenoceptors.

At a time when pharmaceutical companies are having trouble finding new low MW drugs and when biologics are becoming more common, animal venoms could constitute an underexploited source of novel drug candidates. We looked for identifying novel animal toxins active against G protein-coupled receptors (GPCR), the most frequently exploited class of treatment targets, with the aim to develop novel research tools and drug candidates. Screening of green mamba (Dendroaspis angusticeps) venom against adrenoceptors identified two novel venom peptides. ρ-Da1a shown an affinity of 0.35 nM for the α1a-AR while ρ-Da1b displayed affinities between 14 and 73 nM for the three α2-ARs. These two venom peptides have sequences similar to those of muscarinic toxins and belong to the three-finger-fold protein family. α1a-AR is the primary target for the treatment of prostate hypertrophy. In vitro and in vivo tests demonstrated that ρ-Da1a reduced prostatic muscle tone as efficiently as tamsulosin (an antagonist presently used), but with fewer cardiovascular side effects. α2-ARs are the prototype of GPCRs not currently used as treatment targets due to a lack of specific ligands. Blockage of these receptors increases intestinal motility, which may be compromised by abdominal surgery and reduces orthosteric hypotension. In vitro and in vivo tests demonstrated that ρ-Da1b antagonizes α2-ARs in smooth muscles and increased heart rate and blood catecholamine concentrations. These results highlight possible exploitation of ρ-Da1a and ρ-Da1b in important pathologies.

5-Hydroxytryptamine potentiates neurogenic contractions of rat isolated urinary bladder through both 5-HT(7) and 5-HT(2C) receptors.

Serotonin (5-HT) enhances the neurogenic contractile response induced by electrical field stimulation (EFS) in the rat isolated urinary bladder. The aim of this study was to functionally characterize the receptors involved in this effect by using a range of 5-HT receptor subtype selective agonists and antagonists. 5-HT produced a concentration-dependent potentiation of contractile responses to EFS with a pEC(50) value of 6.86±0.24. SB-269970 (0.01, 0.1 and 1µM), a selective 5-HT(7) receptor antagonist, caused a concentration-dependent rightward shift of the 5-HT-induced response. The pA(2) value was 8.16 with a slope of 0.46±0.08. Neither ketanserine nor SB-204741, 5-HT(2A) and 5-HT(2B) receptors antagonists, respectively, affected the concentration-response curve to 5-HT. However, 5-HT response was antagonized by the selective 5-HT(2C) receptor antagonist SB-242084 (0.1 and 1µM). In the presence of 1µM of both antagonists SB-269970 and SB-242084, 5-HT response was almost fully inhibited. 5-CT, a 5-HT(7) receptor agonist, induced a biphasic concentration-dependent potentiation of neurogenic contractions. SB-269970 concentration-dependently antagonized the first phase of 5-CT response with a pA(2) value of 8.77 and a slope not significantly different from unity (0.91±0.11) that suggests a competitive antagonism. WAY-161503, a 5-HT(2C) receptor agonist (0.01-10µM), induced a concentration-dependent potentiation of contractile response to EFS while DOI (a selective 5-HT(2A) agonist) had no effect. SB-242084 (0.1 and 1µM) antagonized the effect of WAY-161503 in a concentration-dependent manner. The current results demonstrate that 5-HT potentiates neurogenic contractions of rat isolated detrusor muscle through both 5-HT(7) and 5-HT(2c) receptors.

Halothane-anesthetized rabbit: a new experimental model to test the effects of besipirdine and duloxetine on lower urinary tract function.

INTRODUCTION: The effects of besipirdine and its main metabolite, HP-748, as well as duloxetine and tomoxetine in the lower urinary tract (LUT) were studied using in vitro and in vivo techniques. MATERIALS AND METHODS: For in vivo studies, besipirdine or duloxetine effects on cystometric parameters and striated sphincter electromyographic (SS-EMG) activity were investigated. On the isolated urethra, norepinephrine (NE) concentration-response curves (CRC) were performed in the presence of besipirdine, duloxetine or tomoxetine. Moreover, CRC to HP-748 were constructed in the absence or presence of prazosin. Potency (pEC(50)) and maximal responses (E(max)) were determined. RESULTS: Besipirdine at 1, 3 and 5 mg/kg intravenously (i.v.) induced a significant increase in SS-EMG activity (250, 273 and 241%, respectively), bladder capacity (172, 197, and 235%, respectively), intercontraction interval (ICI; 208, 242, and 400%, respectively), and residual volume (181, 191, and 236%, respectively). Duloxetine at 2 mg/kg i.v. increased significantly SS-EMG activity (219%), micturition volume (222%), and ICI (205%). In the isolated urethra, besipirdine, tomoxetine and duloxetine significantly displaced to the left the NE CRC. In addition, HP-748 induced contraction of the isolated urethra with a pEC(50) of 5.89 and an E(max) of 37%. CONCLUSIONS: These data support the potential of besipirdine as a new drug for LUT dysfunctions such as stress and mixed urinary incontinence.

Involvement of beta(3)-adrenoceptors in mouse urinary bladder function: role in detrusor muscle relaxation and micturition reflex.

beta(3)-adrenoceptor activation produces relaxation of human urinary bladder smooth muscle (detrusor). Therefore, beta(3)-adrenoceptor agonism is being investigated as a new therapeutic strategy for the treatment of overactive bladder. The aim of the current study was to identify the functional presence of beta(3)-adrenoceptors in mouse isolated urinary bladder using the selective beta(3)-adrenoceptor agonist CL316,243 and antagonists SR59230A and L748,337. The effects of CL316,243 on basal tone, spontaneous activity and electrical field stimulation (EFS)-induced contractions were investigated using in vitro techniques, while the in vivo effects of intravenously administered CL316,243 on the micturition reflex were investigated using cystometry. CL316,243 decreased basal tone (pEC(50)=6.4+/-0.4) as well as spontaneous activity (53+/-7% at 3 microM) and inhibited EFS-induced contractions (pEC(50)=7.0+/-0.2) of the detrusor muscle. The beta(3)-adrenoceptor antagonist SR59230A (1 microM) significantly inhibited the relaxing effects of CL316,243 on basal tone and neurogenic contractions (pA(2)=7.0 and 7.2, respectively). Another beta(3)-adrenoceptor antagonist L748,337 (1-10 microM) significantly blocked the CL316,243-evoked inhibition of neurogenic contractions in a concentration-dependent manner (pK(B)=6.8), while the selective beta(2)-adrenoceptor antagonist ICI118,551(30 nM) had no effect. In anesthetized mice, CL316,243 (0.03 and 0.1 mg/kg, i.v.) significantly increased bladder capacity and threshold pressure without a modification of bladder compliance. Moreover, it induced a significant decrease in the amplitude of both micturition and non-voiding contractions. Based on the current results obtained using the beta(3)-adrenoceptor agonist CL316,243 (as well as various beta-adrenoceptor antagonists), functional beta(3)-adrenoceptors appear to be present in mouse urinary bladder.

Comparative effect of alfuzosin and tamsulosin on the contractile response of isolated rabbit prostatic and iris dilator smooth muscles: possible model for intraoperative floppy-iris syndrome.

PURPOSE: To compare the pharmacologic properties of tamsulosin and alfuzosin in isolated prostatic and iris dilator smooth muscle from pigmented rabbits. SETTING: UROsphere Laboratories, Université Paul Sabatier, Toulouse, France. METHODS: Prostatic and iris dilator smooth muscle strips were placed in organ baths. A concentration-response curve to phenylephrine was compared before and after incubation with tamsulosin or alfuzosin. RESULTS: Both drugs were approximately 30 times less potent in iris dilator than prostatic smooth muscle. In the iris, tamsulosin acted as a competitive antagonist starting at the 0.03 microM concentration (pA(2)=7.96). This is in the same range as the maximum plasma concentration after a 0.4 mg dose of tamsulosin in humans (0.025 microM). The antagonistic effect of alfuzosin in the iris was weaker (calculated mean pA(2) value of 5.63+/-0.19). Concentrations with an equipotent antagonistic effect on rabbit iris dilator muscle (3.0 and 10.0 microM) were approximately 100 to 300 times higher than the maximum plasma concentrations after a 10.0 mg dose of alfuzosin in humans (0.032 microM). CONCLUSIONS: Tamsulosin was more effective than alfuzosin at blocking adrenergic contraction of the iris dilator muscle in pigmented rabbits. Both drugs were less potent in the iris than in the prostate, which suggests that an additional iris receptor could be involved. If valid in humans, our results suggest that attainable plasma concentrations of tamsulosin are able to antagonize iris dilator smooth muscle contraction, whereas those of alfuzosin are not. This could explain the higher frequency of intraoperative floppy-iris syndrome in patients treated with tamsulosin than with alfuzosin.

Involvement of 5-hydroxytryptamine (HT)7 receptors in the 5-HT excitatory effects on the rat urinary bladder.

OBJECTIVE: To investigate the in vitro and in vivo effects of 5-hydroxytryptamine (5-HT) on the rat urinary bladder and to characterize the receptors involved in mediating these pharmacological effects by using selective antagonists. MATERIALS AND METHODS: Female Wistar rats (250-350 g) were used for all studies. In vitro, detrusor muscle strips were mounted between two platinum electrodes in organ baths filled with a modified Krebs' solution bubbled with 95% O(2) and 5% CO(2) at 37 degrees C. After equilibration and a contraction to 80 mmol/L KCl, strips were exposed to electrical field stimulation for 30 min and incubated with the antagonist or vehicle for a further 30 min, then a 5-HT concentration-response curve (CRC) was obtained. In vivo, rats were anaesthetized with pentobarbital, and the ureters and urethra ligated, the bladder catheterized and infused with saline. 5-HT (3-100 microg/kg intravenous) dose-dependently increased intravesical pressure (IVP). After administering 5-HT at 30 microg/kg three times at 10 min intervals (controls), one dose of antagonist was perfused for 5 min and, after a further 5 min, 30 microg/kg 5-HT was tested again. This cycle was repeated four times using increasing doses of the antagonist to be tested. RESULTS: In vitro, 5-HT (0.01-100 micromol/L) induced a concentration-dependent enhancement of the neurogenic response, with a mean (sd) pEC(50) of 6.36 (0.15) and E(max) of 41.1 (4.6)% KCl (eight rats). In unstimulated tissues, 5-HT induced no contractile effect. Selective 5-HT(4), 5-HT(3) and 5-HT(1A) receptor antagonists had no effect on the 5-HT potentiating effects. The potentiating effect of 5-HT was antagonized by mesulergine at 0.3 micromol/L, R(+)lisuride at 0.3 micromol/L and the selective 5-HT(7) receptor antagonist SB-258741 at 0.3 micromol/L. In vivo, in anaesthetized rats, IVP increases induced by repeated doses of 30 microg/kg 5-HT were reproducible. R(+)lisuride (3-100 microg/kg) dose-dependently inhibited the 5-HT-induced increase of IVP. At the maximum dose tested, R(+)lisuride almost totally inhibited the 5-HT effect. CONCLUSIONS: In rat isolated detrusor muscle the 5-HT(7) receptor antagonists SB-258741, R(+)lisuride and mesulergine blocked the 5-HT potentiating effect with the expected potency. Moreover, in anaesthetized rats, R(+)lisuride abolished 5-HT effects on IVP at doses that antagonize physiological effects known to be mediated by 5-HT(7) receptor activation in several animal species. These results suggest the involvement of 5-HT(7) receptors in the modulation of rat bladder contraction both in vitro and in vivo.

Cholinergic and purinergic contribution to the micturition reflex in conscious rats with long-term bladder outlet obstruction.

The urethra of female Wistar rats was partially obstructed for 15 weeks. The effects of atropine (1 mg/kg i.v.), suramin (100 mg/kg i.v.), and a combination of atropine and suramin on the peak micturition pressure (MP) were compared during cystometry in conscious rats controls or subjected to outlet obstruction. On the isolated bladder dome, we studied the inhibitory effect of 1 micromol/L atropine, 1 mmol/L suramin, and the combination of the two drugs on contractions induced by electrical field stimulation (EFS). We studied also the contractile response to 80 mmol/L KCl and the concentration-response curves to noradrenaline, phenylephrine, and carbachol on the bladder dome and bladder neck and alpha, beta-methylene adenosine triphosphate on the bladder dome. In conscious rats, the MP, bladder capacity, and micturition volume were significantly higher in obstructed rats than in controls. Suramin induced the same inhibition in the two groups of animals (-30.7 +/- 13.3% in controls and -29.2 +/- 8.5% in obstructed rats). Atropine decreased the MP, but this effect was twofold greater in obstructed animals (-28.1 +/- 3.1% and -65.1 +/- 6.9% in control and obstructed animals, respectively). However, the combined effect of atropine and suramin was additive in controls but not in obstructed (-56.7 +/- 5.4% and -55.9 +/- 9.4%, respectively). Similar results were obtained in vitro using 1 micromol/L atropine and 1 mmol/L suramin. In the obstructed bladder dome and bladder neck, we found a great reduction in KCl- and carbachol-induced contractility but no difference in the response to EFS. Responses to noradrenaline and phenylephrine were moderately reduced in the bladder neck only, whereas responses to alpha, beta-methylene adenosine triphosphate in the bladder dome were not reduced except at the concentration of 300 micromol/L. We conclude that long-term obstruction in rats could induce cholinergic nerve fiber proliferation as suggested by the decrease in M(3) muscarinic receptor contractility (desensitization) and by a greater sensitivity of the MP to atropine.