Comparison of the effects of ß3 -adrenoceptor agonism on urinary bladder function in conscious, anesthetized, and spinal cord injured rats.

AIMS: To compare the dose effect relationship of a selective ß3 -adrenoceptor agonist (CL-316,243) on cystometric parameters in anesthetized and conscious rats and to evaluate its effect in a model of neurogenic bladder overactivity induced by spinal cord injury (SCI). METHODS: Experiments were performed in anesthetized and conscious normal rats and in conscious rats after complete transection at the T8 level of the spinal cord. The jugular vein and urinary bladder were catheterized and the bladder infused with saline. CL-316,243 was tested intravenously at 0.01, 0.03, and 0.1 mg/kg in anesthetized and conscious rats and at 0.01 mg/kg in sham and SCI rats. Intravesical pressure was recorded for 1 hr following drug administration. Intercontraction interval (ICI), amplitude of micturition (AM), micturition frequency (MF) and non-voiding contractions (NVC) were analyzed. RESULTS: In anesthetized and conscious normal rats, CL-316,243 significantly increased ICI in a dose-dependent manner. In anesthetized rats, AM was significantly decreased at all doses tested whereas in conscious rats, a significant decrease (-19 ± 6%) in AM was only observed at the highest dose (0.1 mg/kg). In conscious sham and SCI rats, CL-316,243 significantly increased ICI (42 ± 17% and 49 ± 17%, respectively) and decreased MF without affecting AM. In SCI rats, CL-316,243 reduced the frequency of NVC (-53 ± 14%) without significant effects on amplitude. CONCLUSIONS: The current results suggest that anesthesia can alter the effects of ß3 -adrenoceptor agonists in experimental models. In addition, this is the first demonstration that stimulation of ß3 -adrenoceptors can produce decreases in micturition frequency and NVC in SCI rats without affecting AM.

ADX71441, a novel, potent and selective positive allosteric modulator of the GABA(B) receptor, shows efficacy in rodent models of overactive bladder.

BACKGROUND AND PURPOSE: The GABAB receptor agonist baclofen reduces urethral resistance and detrusor overactivity in patients with spasticity. However, baclofen's side effects limit its use for the treatment of overactive bladder (OAB). Here, we tested a novel GABAB positive allosteric modulator (PAM) ADX71441 in models of OAB in mice and guinea pigs. EXPERIMENTAL APPROACH: Mice were left untreated or given (p.o.) vehicle (1% CMC), ADX71441 (1, 3, 10 mg kg(-1) ) or oxybutynin (100 mg kg(-1) ; Experiment 1) or vehicle (1% CMC), baclofen (1, 3, 6 mg kg(-1) ) or oxybutynin (Experiment 2). Treated mice were then overhydrated with water, challenged with furosemide, before being placed into micturition chambers and monitored for urinary parameters. In anaesthetized guinea pigs, intravesical infusion of acetic acid was used to induce OAB and the effects of ADX71441 (1, 3 mg kg(-1) ) or baclofen (1 mg kg(-1) ), administered i.v., on cystometric parameters were monitored. KEY RESULTS: In mice, 10 mg kg(-1) ADX71441 increased urinary latencies, reduced the number of urinary events and the total and average urinary volumes. In guinea pigs, ADX71441 (1 and 3 mg kg(-1) ) increased the intercontraction interval (ICI) and bladder capacity (BC), and reduced micturition frequency (MF) compared to vehicle. At 3 mg kg(-1) ADX71441 completely inhibited the micturition reflex and induced overflow incontinence in five out of 10 animals. Baclofen slightly increased ICI and BC and reduced MF. CONCLUSION AND IMPLICATIONS: Our findings demonstrate, for the first time, that a GABAB PAM has potential as a novel approach for the treatment of OAB.

Effects of ρ-Da1a a peptidic α(1) (A) -adrenoceptor antagonist in human isolated prostatic adenoma and anaesthetized rats.

BACKGROUND AND PURPOSE: ρ-Da1a, a 65 amino-acid peptide, has subnanomolar affinity and high selectivity for the human α(1) (A) -adrenoceptor subtype. The purpose of this study was to characterize the pharmacological effects of ρ-Da1a on prostatic function, both in vivo and in vitro. EXPERIMENTAL APPROACH: ρ-Da1a was tested as an antagonist of adrenaline-induced effects on COS cells transfected with the human α(1) (A) -adrenoceptor as well as on human isolated prostatic adenoma obtained from patients suffering from benign prostatic hyperplasia. Moreover, we compared the effects of ρ-Da1a and tamsulosin on phenylephrine (PHE)-induced increases in intra-urethral (IUP) and arterial pressures (AP) in anaesthetized rats, following i.v. or p.o. administration. KEY RESULTS: On COS cells expressing human α(1) (A) -adrenoceptors and on human prostatic strips, ρ-Da1a inhibited adrenaline- and noradrenaline-induced effects. In anaesthetized rats, ρ-Da1a and tamsulosin administered i.v. 30 min before PHE significantly antagonized the effects of PHE on IUP. The pK(B) values for tamsulosin and ρ-Da1a for this effect were similar. With regards to AP, ρ-Da1a only reduced the effect of PHE on AP at the lowest dose tested (10 µg·kg(-1) ), whereas tamsulosin significantly reduced PHE effects at doses between 10 and 150 µg·kg(-1) . CONCLUSIONS AND IMPLICATIONS: ρ-Da1a exhibited a relevant effect on IUP and a small effect on AP. In contrast, tamsulosin antagonized the effects of PHE on both IUP and AP. We conclude that ρ-Da1a is more uroselective than tamsulosin. ρ-Da1a is the most selective peptidic antagonist for α(1A) -adenoceptors identified to date and could be a new treatment for various urological diseases.

Isolation and pharmacological characterization of AdTx1, a natural peptide displaying specific insurmountable antagonism of the alpha1A-adrenoceptor.

BACKGROUND AND PURPOSE: Venoms are a rich source of ligands for ion channels, but very little is known about their capacity to modulate G-protein coupled receptor (GPCR) activity. We developed a strategy to identify novel toxins targeting GPCRs. EXPERIMENTAL APPROACH: We studied the interactions of mamba venom fractions with alpha(1)-adrenoceptors in binding experiments with (3)H-prazosin. The active peptide (AdTx1) was sequenced by Edman degradation and mass spectrometry fragmentation. Its synthetic homologue was pharmacologically characterized by binding experiments using cloned receptors and by functional experiments on rabbit isolated prostatic smooth muscle. KEY RESULTS: AdTx1, a 65 amino-acid peptide stabilized by four disulphide bridges, belongs to the three-finger-fold peptide family. It has subnanomolar affinity (K(i)= 0.35 nM) and high specificity for the human alpha(1A)-adrenoceptor subtype. We showed high selectivity and affinity (K(d)= 0.6 nM) of radio-labelled AdTx1 in direct binding experiments and revealed a slow association constant (k(on)= 6 x 10(6).M(-1).min(-1)) with an unusually stable alpha(1A)-adrenoceptor/AdTx1 complex (t(1/2diss)= 3.6 h). AdTx1 displayed potent insurmountable antagonism of phenylephrine's actions in vitro (rabbit isolated prostatic muscle) at concentrations of 10 to 100 nM. CONCLUSIONS AND IMPLICATIONS: AdTx1 is the most specific and selective peptide inhibitor for the alpha(1A)-adrenoceptor identified to date. It displays insurmountable antagonism, acting as a potent relaxant of smooth muscle. Its peptidic nature can be exploited to develop new tools, as a radio-labelled-AdTx1 or a fluoro-labelled-AdTx1. Identification of AdTx1 thus offers new perspectives for developing new drugs for treating benign prostatic hyperplasia.

New metabolic and pharmacokinetic characteristics of thiocolchicoside and its active metabolite in healthy humans.

Thiocolchicoside (TCC) has been prescribed for several years as a muscle relaxant drug, but its pharmacokinetic (PK) profile and metabolism still remain largely unknown. Therefore, we re-investigated its metabolism and PK, and we assessed the muscle relaxant properties of its metabolites. After oral administration of 8 mg (a therapeutic dose) of 14C-labelled TCC to healthy volunteers, we found no detectable TCC in plasma, urine or faeces. On the other hand, the aglycone derivative obtained after de-glycosylation of TCC (M2) was observed and, in addition, we identified, as the major circulating metabolic entity, 3-O-glucuronidated aglycone (M1) obtained after glucuro-conjugation of M2. One hour after oral administration, M1 plus M2 accounted for more than 75% of the circulating total radioactivity. The pharmacological activity of these metabolites was assessed using a rat model, the muscle relaxant activity of M1 was similar to that of TCC whereas M2 was devoid of any activity. Subsequently, to investigate the PK profile of TCC in human PK studies, we developed and validated a specific bioanalytical method that combines liquid chromatography and ultraviolet detection to assay both active entities. After oral administration, TCC was not quantifiable with an lower limit of quantification set at 1 ng/mL, whereas its active metabolite M1 was detected. M1 appeared rapidly in plasma (tmax=1 h) and was eliminated with an apparent terminal half-life of 7.3 h. In contrast, after intramuscular administration both active entities (TCC and M1) were present; TCC was rapidly absorbed (tmax=0.4 h) and eliminated with an apparent terminal half-life of 1.5 h. M1 concentration peaked at 5 h and this metabolite was eliminated with an apparent terminal half-life of 8.6 h. As TCC and M1 present an equipotent pharmacological activity, the relative oral pharmacological bioavailability of TCC vs. intramuscular administration was calculated and represented 25%. Therefore, to correctly investigate the PK and bioequivalence of TCC, the biological samples obtained must be assayed with a bioanalytical method able to specifically analyse TCC and its active metabolite M1.

Age-related changes in urethrovesical coordination in male rats: relationship with bladder instability?

The micturition profile in conscious animals and the urethrovesical coordination in anesthetized conditions were investigated in 6- and 24-mo-old male Sprague-Dawley rats. The in vitro pharmacological responses to KCl, electrical field stimulation (EFS), carbachol, phenylephrine, and isoprenaline were determined in the isolated bladder body, the bladder neck, and urethra. A morphometric and immunohistological study has been included. During conscious cystomanometry, 63% of the aging rats but only 25% of the adult rats showed spontaneous contractions during the bladder-filling phase. In conscious aging rats, basal pressure, threshold pressure, and micturition pressure were also significantly increased. In anesthetized aging rats, a decrease in resting urethral pressure at micturition threshold and the occurrence of a significant delay in urethral relaxation during micturition were associated with an increased residual volume. In all isolated tissues, contractile response to KCl was not modified with aging, whereas age-related decreases in maximal responses to carbachol in the bladder body and to phenylephrine and carbachol in the urethra were observed. In the bladder neck only, we found a significant decrease in the amplitude of neurogenic contractions associated with fibrosis but without decrease in nerve density. These experiments show significant modifications in the voiding pattern of aging rats associated with urethral dysfunction and with regionally specific pharmacological and structural changes of the urinary tract. We propose that aging in rats is characterized by an impairment of the urethrovesical coordination, leading to bladder dysfunctions similar to those induced by bladder outlet obstruction.

Increased adrenergic contractility and decreased mRNA expression of NOS III in aging rat urinary bladders.

Our objective was to study age-related changes in adrenergic contractility and gene expression profile in the rat urinary bladder. Young (3-month old), adult (10-month old) and senescent (30-month old) male WAG/Rij rats were used. Gene expression profile in the rat urinary bladder was defined using Atlas microarray technology. In vitro contractile responses induced by KCl, phenylephrine (PHE) and norepinephrine (NE) were compared in isolated urinary bladders dissected from young, adult and senescent rats. Among a total of 1176 genes present on the arrays, 15 genes showed an increase in expression and 10 genes a decrease with age. Four genes related to nerve growth factor were upregulated whereas NOS type III was downregulated in aging rats. Intrinsic contractility of isolated rat urinary bladders was not changed between adult and aging rats as judged by the response curves to KCl. In contrast, an age-related increase in the maximal contractile responses to NE, but not PHE, was noticed (13 +/- 1, 48 +/- 2% and 59 +/- 2% at 3, 10 and 30 months, respectively). The alpha1D-adrenoceptor antagonist BMY7378 antagonized NE-induced contractions with low potency in both groups suggesting the involvement of the alpha1A-adrenoceptor subtype. This was confirmed by microarray, which demonstrated mRNA expression for the alpha1A-adrenoceptor subtype only. These results suggest that aging of the urinary bladder is associated with an increase in the maximal contractile response to NE which could be due to NO shortage resulting from downregulation of urothelial NOS III.

Functional and morphological modifications of the urinary bladder in aging female rats.

In female Wistar/Rij rats, 10 and 30 mo old, the micturition profiles in conscious animals, the contractile responses of the isolated urinary bladder, and the histology of the vesical tissue have been investigated. During cystomanometry, 60% of conscious senescent rats, but only 25% of young adult rats, showed spontaneous contractions during the bladder-filling phase. In aging rats, micturition pressure and duration of micturition were significantly higher by approximately 40-50%. In contrast, bladder capacity, bladder compliance, micturition volume, and residual volume were not modified with age. In vitro, the contractile responses of the bladder body to KCl, carbachol, arecoline, and alpha,beta-MeATP were similar in tissues from young adult and senescent rats. In contrast, maximum responses to noradrenaline, but not phenylephrine, were two times greater in the older rats. Isoprenaline exhibited the same potency in relaxing KCl-precontracted bladder body of 10- and 30-mo-old animals. Morphometric analysis showed a significant increase in the mean thickness of the muscularis layer with age, whereas the collagen density significantly decreased in the muscularis and in the lamina propria layers. The fact that the majority of senescent rats displayed bladder instability and increased response to alpha-adrenergic agonists suggests that this strain of rat seems a good model for the aged human. However, other characteristics of the aging human urinary tract (urinary frequency, decreased cystometric capacity, and decreased detrusor contractility associated with fibrosis) are not present.

Pharmacological and urodynamic changes in rat urinary bladder function after multiple pregnancies.

OBJECTIVE: To investigate the changes in bladder function after multiple pregnancies and parturition in rats, and to establish links between the changes in voiding profiles and in vitro pharmacological responses. MATERIALS AND METHODS: Cystometry was used in conscious virgin and multiparous female Wistar rats (2 weeks after the last parturition) with chronically implanted lines to continuously record bladder pressure during five reproducible voiding cycles. In vitro, detrusor muscle contractile responses induced by cumulative concentrations of KCl, carbachol, noradrenaline and alpha, beta-methylene-ATP (mATP) were compared. RESULTS: In multiparous rats, there was a significant increase in the amplitude of voiding pressure (+31%), bladder capacity (+83%) and residual volume (about threefold); 60% of the multiparous rats but only 10% of the virgin rats showed bladder instability during the filling phase. Cumulative concentration-response curves to KCl, expressed as the tension developed vs tissue weight, were identical in the two groups of rats. Contractile responses induced by carbachol (0.1-30 micromol/L) were significantly larger in multiparous than in virgin rats. Similarly, noradrenaline-induced contractions (0.3-10 micromol/L) were significantly higher for multiparous animals. However, the sensitivity of the detrusor muscle to mATP was not modified by multiple pregnancies. CONCLUSION: After multiple gestations, female rats develop bladder hypertrophy, bladder instabilities and a higher amplitude of voiding pressure associated with an increased residual volume. These altered patterns are similar to those found in rats after chronic infravesical outlet obstruction. We propose that pregnancies and parturition modify urinary bladder function, leading to a dysfunction similar to that induced by obstruction, and involving an increased sensitivity to adrenergic and cholinergic stimulation.

Experimental bladder instability following bladder outlet obstruction in the female rat.

PURPOSE: In rats, urethral obstruction was previously shown to lead to detrusor instability, but bladder instability in conscious animals has not been well characterized. The present study was designed to describe the consequences of bladder outlet obstruction on urodynamic parameters in the conscious rat. A categorization of different types of bladder instability in conscious rats is described. MATERIALS AND METHODS: Cystometry was performed on conscious animals with chronically implanted lines. Recordings of the micturition pattern and the bladder pressure during the micturition cycle were made from normal female rats and those with instability caused by infravesical outflow obstruction. RESULTS: In control rats, the peak micturition pressure was low (45.6+/-2.5 cm. H2O) and only small pressure fluctuations occurred before or after micturition. Rats with outlet obstruction exhibited bladder hypertrophy, high micturition pressure (95.5+/-6.8 cm. H2O) as well as marked spontaneous activity throughout the micturition cycle. According to the spontaneous activity profile we identified four main patterns of abnormal micturition cycles. CONCLUSION: Urethral obstruction in rats leads to a significant increase in bladder weight and to altered micturition patterns which reveal clear similarities with features of the cystometric profiles performed in men with infravesical outflow obstruction. These modifications are not consistently correlated with an increase in bladder weight.

Relationship between the effects of alfuzosin on rat urethral and blood pressures and its tissue concentrations.

This study was undertaken in order to establish the alpha1-antagonist effects of alfuzosin on phenylephrine-induced increases in urethral and arterial blood pressures at 1 and 6 hours post dosing (10 mg/kg, p.o.). At each time, plasma and prostatic concentrations of alfuzosin were measured and correlations between tissue concentrations and pharmacological effects were calculated. At one and six hours post dosing, alfuzosin markedly shifted the urethral and arterial dose response curve to phenylephrine. At one hour, prostatic concentration was 4.1 times greater than plasma concentration (363 ng/g vs 88 ng/ml) and at 6 hours this ratio reached 8.6 times (167 ng/g vs 20 ng/ml). By taking together the data points obtained at 1 and 6 hours we showed that the effects of alfuzosin on urethral pressure were correlated with prostate levels (r=0.906, p<0.01) and the effects on arterial blood pressure were correlated with plasma levels (r=0.941, p<0.01). These results suggest that a preferential distribution of alfuzosin in prostatic tissue may play a role in its functional uroselectivity.

Comparative alpha-1 adrenoceptor subtype selectivity and functional uroselectivity of alpha-1 adrenoceptor antagonists.

We investigated the relevance of selectivity for a given alpha-1-adrenoceptor subtype for in vivo uroselectivity of several alpha-1-adrenoceptor antagonists (alfuzosin, doxazosin, prazosin, tamsulosin, terazosin and 5-Me-urapidil). Comparison of the affinities of these alpha-1-adrenoceptor antagonists at the cloned alpha-1a, alpha-1b and alpha-1d-adrenoceptor subtypes revealed that tamsulosin and 5-Me-urapidil showed selectivity for the alpha-1a subtype. No significant correlations were found between the affinities for alpha-1b or alpha-1d-adrenoceptors and the pK(B) values obtained against phenylephrine-induced contraction of the rabbit prostate in vitro. In contrast, the antagonist potencies in rabbit prostate were correlated (r = 0.89, P < .05) with the pKi values for the alpha-1a-adrenoceptor subtype. However, the pK(B) values were consistently smaller (by 0.6 to 1.9 log unit) than the pKi values for the alpha-1a-adrenoceptor subtype, a result that suggests that the alpha-1-adrenoceptor mediating urethral contractions does not have all the characteristics of the alpha-1a-adrenoceptor. The simultaneous measurement of urethral and arterial pressures in the same conscious male rat allows evaluation of the functional uroselectivity of these antagonists based on their respective effects on both pressures. Dose ranges were selected according to effects on urethral pressure and most antagonists were found effective within the 3 to 100 microg/kg i.v. range. Alfuzosin markedly decreased urethral pressure and either did not decrease blood pressure (10-30 microg/kg) or slightly decreased it at the highest dose tested (100 microg/kg). Doxazosin did not produce sustained reductions in urethral pressure until a dose of 30 microg/kg. Blood pressure was not reduced until 100 microg/kg. Prazosin reduced urethral pressure and blood pressure within the same dose-range whereas terazosin did not decrease urethral pressure at doses that significantly decreased blood pressure (30 and 100 microg/kg). 5-Me-urapidil, an alpha-1a-selective compound did not significantly modify urethral and blood pressure whereas tamsulosin, another alpha-1a-selective compound reduced urethral pressure and blood pressure within the same dose range. In conclusion, in the conscious male rat the functional uroselectivity is not correlated with a selective affinity for the alpha-1a-adrenoceptor subtype.

Vasopressin V2 receptor mRNA expression and cAMP accumulation in aging rat kidney.

The ability of the kidney to regulate water balance is impaired with age, although the secretion of vasopressin is maintained in senescent animals. This suggests that the cellular response to antidiuretic hormone is reduced in aging kidney. To test this hypothesis, the relationship between the expression of the vasopressin. V2 receptor mRNA and adenosine 3',5'-cyclic monophosphate (cAMP) accumulation was investigated in the medullary thick ascending limb of Henle's loop (MTAL) of adult and aging rats. Tubular suspensions of MTAL were prepared from 10- and 30-mo-old female WAG/Rij rats. The accumulation of cAMP for maximal concentration of vasopressin was 34% larger in adult than in old animals (9.5 +/- 0.5 pmol/4 min, n = 16, and 7.1 +/- 0.6 pmol/4 min, n = 12, respectively). The concentration of vasopressin corresponding to half-maximal stimulation was similar in the two groups (0.66 +/- 0.20 and 0.52 +/- 0.09 nmol, n = 5, in adult and old animals), indicating comparable sensitivity of the renal cells with age. The age-related impaired response to vasopressin of the V2 receptor was specific for females and was not observed in males. Direct stimulation of adenylyl cyclase by forskolin induced a comparable accumulation of cAMP in adult and senescent rats. The V2 receptor mRNA level in the MTAL was constant between 10 and 30 mo whether the animals were normally hydrated or dehydrated for 2 days. These data indicate that, in MTAL, the age-related impaired cAMP accumulation by vasopressin would be linked to a change either in the translation of V2 mRNA or in posttranslational processing mechanisms or in the coupling between the V2 receptor and adenylyl cyclase.