Effects of fatty acid metabolites on nocturia.

Dysregulation of circadian rhythm can cause nocturia. Levels of fatty acid metabolites, such as palmitoylethanolamide (PEA), 9-hydroxy-10E,12Z-octadecadienoic acid (9-HODE), and 4-hydroxy-5E,7Z,10Z,13Z,16Z,19Z-docosahexaenoic acid (4-HDoHE), are higher in the serum of patients with nocturia; however, the reason remains unknown. Here, we investigated the circadian rhythm of fatty acid metabolites and their effect on voiding in mice. WT and Clock mutant (ClockΔ19/Δ19) mice, a model for nocturia with circadian rhythm disorder, were used. Levels of serum PEA, 9-HODE, and 4-HDoHEl were measured every 8 h using LC/MS. Voiding pattern was recorded using metabolic cages after administration of PEA, 9-HODE, and 4-HDoHE to WT mice. Levels of serum PEA and 9-HODE fluctuated with circadian rhythm in WT mice, which were lower during the light phase. In contrast, circadian PEA and 9-HODE level deteriorated or retreated in ClockΔ19/Δ19 mice. Levels of serum PEA, 9-HODE, and 4-HDoHE were higher in ClockΔ19/Δ19 than in WT mice. Voiding frequency increased in PEA- and 4-HDoHE-administered mice. Bladder capacity decreased in PEA-administered mice. The changes of these bladder functions in mice were similar to those in elderly humans with nocturia. These findings highlighted the novel effect of lipids on the pathology of nocturia. These may be used for development of biomarkers and better therapies for nocturia.

Urothelium-Specific Deletion of Connexin43 in the Mouse Urinary Bladder Alters Distension-Induced ATP Release and Voiding Behavior.

Connexin43 (Cx43), the main gap junction and hemichannel forming protein in the urinary bladder, participates in the regulation of bladder motor and sensory functions and has been reported as an important modulator of day-night variations in functional bladder capacity. However, because Cx43 is expressed throughout the bladder, the actual role played by the detrusor and the urothelial Cx43 is still unknown. For this purpose, we generated urothelium-specific Cx43 knockout (uCx43KO) mice using Cre-LoxP system. We evaluated the day-night micturition pattern and the urothelial Cx43 hemichannel function of the uCx43KO mice by measuring luminal ATP release after bladder distention. In wild-type (WT) mice, distention-induced ATP release was elevated, and functional bladder capacity was decreased in the animals' active phase (nighttime) when Cx43 expression was also high compared to levels measured in the sleep phase (daytime). These day-night differences in urothelial ATP release and functional bladder capacity were attenuated in uCx43KO mice that, in the active phase, displayed lower ATP release and higher functional bladder capacity than WT mice. These findings indicate that urothelial Cx43 mediated ATP signaling and coordination of urothelial activity are essential for proper perception and regulation of responses to bladder distension in the animals' awake, active phase.

Social isolation uncovers a circuit underlying context-dependent territory-covering micturition.

The release of urine, or micturition, serves a fundamental physiological function and, in many species, is critical for social communication. In mice, the pattern of urine release is modulated by external and internal factors and transmitted to the spinal cord via the pontine micturition center (PMC). Here, we exploited a behavioral paradigm in which mice, depending on strain, social experience, and sensory context, either vigorously cover an arena with small urine spots or deposit urine in a few isolated large spots. We refer to these micturition modes as, respectively, high and low territory-covering micturition (TCM) and find that the presence of a urine stimulus robustly induces high TCM in socially isolated mice. Comparison of the brain networks activated by social isolation and by urine stimuli to those upstream of the PMC identified the lateral hypothalamic area as a potential modulator of micturition modes. Indeed, chemogenetic manipulations of the lateral hypothalamus can switch micturition behavior between high and low TCM, overriding the influence of social experience and sensory context. Our results suggest that both inhibitory and excitatory signals arising from a network upstream of the PMC are integrated to determine context- and social-experience-dependent micturition patterns.

Bladder outlet obstruction disrupts circadian bladder function in mice.

The circadian clock programs daily rhythms and coordinates multiple behavioural processes, including micturition. Partial bladder outlet obstruction (pBOO) in mice produces hyperactive voiding. However, long-term effects of pBOO on bladder function have not been clarified. In this study, we investigated micturition under conditions of impaired circadian bladder function by inducing long-term pBOO by tying the proximal urethra. Micturition behavior was evaluated at 1, 3, 6 and 12 months after surgery. We used automated voided stain on paper method for a precise micturition recording for mice. And quantitative assessment of gene expression was performed at 24 months after pBOO surgery using qRT-PCR procedure. The micturition frequencies in the pBOO group were significantly decreased at 3, 6, and 12 months compared to those at 1 month after operation in the same group (p < 0.05). Body weight of pBOO mice was significantly increased compared to sham operated mice at 12 months. The expression level of mRNA was exhibited a 3.4-fold nominal increased for a 5-HT2B receptor in the pBOO group compared to the sham group. The current study found that long-term pBOO led to disruption of the circadian bladder function (the day/night cycle) in mice, similar to those observed in human as nocturia. This disruption is possible involvement of the gain of body weight and/or serotonergic alteration after pBOO.

Mouse urothelial genes associated with voiding behavior changes after ovariectomy and bladder lipopolysaccharide exposure.

AIMS: Symptoms from overactive bladder (OAB) and cystitis secondary to urinary tract infection (UTI) can be similar in post-menopausal women. Effects of ovariectomy (OVX) on voiding behavior after lipopolysaccharide (LPS) intravesical exposure (surrogate for cystitis) in mice were measured. Urothelial genes associated with micturition changes were identified. METHODS: Female C57BL6/J mice underwent OVX or sham surgeries (n = 10 for each). Voiding spot assays (VSA) were performed prior to surgery, 4 weeks post-surgery, and each time after 3 consecutive days of transurethral instillation of LPS. In another experiment, mice underwent either sham (n = 9) or OVX (n = 9) surgeries. Urothelial RNAs were collected 4 weeks post-surgery, day 1 and day 3 after LPS instillation. Mouse Gene 2.0 ST Arrays (entire 34 K transcripts) were used for microarray hybridization. A set of criteria was utilized to identify gene expression changes that mimicked voiding behavior changes. RESULTS: Three days after LPS exposure, OVX mice persisted with overactive whereas sham mice normalized voiding behavior. Nine urothelial paralleling voiding behavior changes were identified: IL6 (interleukin 6), IL6rα (Interleukin 6 receptor α), Ptgs2 (Prostaglandin-endoperoxide synthase 2 or COX-2), Ereg (epiregulin), Dusp6 (dual specificity phosphatase 6), Zfp948 (zinc finger protein 948), Zfp52 (Zinc finger protein 52), Gch1 (GTP cyclohydrolase 1), and Amd (S-adenosylmethionine decarboxylase). Three other genes, coding unknown proteins, were also identified: GM12840, GM23134, and GM26809. CONCLUSIONS: OVX mice persisted with increased voiding frequency after LPS. Urothelial genes that could mediate this voiding behavior include IL6, COX-2, and S-adenosylmethionine decarboxylase.

Machine Learning on a Genome-wide Association Study to Predict Late Genitourinary Toxicity After Prostate Radiation Therapy.

PURPOSE: Late genitourinary (GU) toxicity after radiation therapy limits the quality of life of prostate cancer survivors; however, efforts to explain GU toxicity using patient and dose information have remained unsuccessful. We identified patients with a greater congenital GU toxicity risk by identifying and integrating patterns in genome-wide single nucleotide polymorphisms (SNPs). METHODS AND MATERIALS: We applied a preconditioned random forest regression method for predicting risk from the genome-wide data to combine the effects of multiple SNPs and overcome the statistical power limitations of single-SNP analysis. We studied a cohort of 324 prostate cancer patients who were self-assessed for 4 urinary symptoms at 2 years after radiation therapy using the International Prostate Symptom Score. RESULTS: The predictive accuracy of the method varied across the symptoms. Only for the weak stream endpoint did it achieve a significant area under the curve of 0.70 (95% confidence interval 0.54-0.86; P = .01) on hold-out validation data that outperformed competing methods. Gene ontology analysis highlighted key biological processes, such as neurogenesis and ion transport, from the genes known to be important for urinary tract functions. CONCLUSIONS: We applied machine learning methods and bioinformatics tools to genome-wide data to predict and explain GU toxicity. Our approach enabled the design of a more powerful predictive model and the determination of plausible biomarkers and biological processes associated with GU toxicity.

Accelerated onset of the vesicovesical reflex in postnatal NGF-OE mice and the role of neuropeptides.

The mechanisms underlying the postnatal maturation of micturition from a somatovesical to a vesicovesical reflex are not known but may involve neuropeptides in the lower urinary tract. A transgenic mouse model with chronic urothelial overexpression (OE) of NGF exhibited increased voiding frequency, increased number of non-voiding contractions, altered morphology and hyperinnervation of the urinary bladder by peptidergic (e.g., Sub P and CGRP) nerve fibers in the adult. In early postnatal and adult NGF-OE mice we have now examined: (1) micturition onset using filter paper void assays and open-outlet, continuous fill, conscious cystometry; (2) innervation and neurochemical coding of the suburothelial plexus of the urinary bladder using immunohistochemistry and semi-quantitative image analyses; (3) neuropeptide protein and transcript expression in urinary bladder of postnatal and adult NGF-OE mice using Q-PCR and ELISAs and (4) the effects of intravesical instillation of a neurokinin (NK)-1 receptor antagonist on bladder function in postnatal and adult NGF-OE mice using conscious cystometry. Postnatal NGF-OE mice exhibit age-dependent (R2=0.996-0.998; p≤0.01) increases in Sub and CGRP expression in the urothelium and significantly (p≤0.01) increased peptidergic hyperinnervation of the suburothelial nerve plexus. By as early as P7, NGF-OE mice exhibit a vesicovesical reflex in response to intravesical instillation of saline whereas littermate WT mice require perigenital stimulation to elicit a micturition reflex until P13 when vesicovesical reflexes are first observed. Intravesical instillation of a NK-1 receptor antagonist, netupitant (0.1µg/ml), significantly (p≤0.01) increased void volume and the interval between micturition events with no effects on bladder pressure (baseline, threshold, peak) in postnatal NGF-OE mice; effects on WT mice were few. NGF-induced pleiotropic effects on neuropeptide (e.g., Sub P) expression in the urinary bladder contribute to the maturation of the micturition reflex and are excitatory to the micturition reflex in postnatal NGF-OE mice. These studies provide insight into the mechanisms that contribute to the postnatal development of the micturition reflex.

Evaluation of voiding assays in mice: impact of genetic strains and sex.

Void spot assays (VSA) and cystometry are two of the most common tests performed in mice to assess lower urinary tract function. Assay protocols and methodology vary greatly among laboratories, and little is known about reproducibility of results generated by different laboratories. We performed VSA in four mouse strains, comparing males with females and comparing results between two independent laboratories. Unique aspects of the current study include direct comparison of results of VSA performed in a similar manner in two locations and comparison of cystometry performed using two different rates of infusion in these two laboratories. Both assays were performed in male and female 129S1/SvImJ, C57BL/6J, NOD/ShiLtJ, and CAST/EiJ mice, and cystometry was performed under urethane anesthesia (10/group). Assays were performed and results analyzed as previously described. Results obtained in female mice were compared with previously reported values. Results of lower urinary tract function testing in mice vary in a consistent manner with strain and sex. Variables in husbandry, testing techniques, and analysis of results can significantly affect conclusions, particularly those obtained by cystometry. Although VSA results were remarkably similar between the two laboratories, consistent methods for performing lower urinary tract function testing in mice are required to compare results among studies with confidence.

Caffeine enhances micturition through neuronal activation in micturition centers.

Caffeine may promote incontinence through its diuretic effect, particularly in individuals with underlying detrusor overactivity, in addition to increasing muscle contraction of the bladder smooth muscle. Caffeine may also affect bladder function via central micturition centers, including the medial preoptic area, ventrolateral periaqueductal gray, and pontine micturition center. However, the biochemical mechanisms of caffeine in central micturition centers affecting bladder function remain unclear. In the present study, the effects of caffeine on the central micturition reflex were investigated by measuring the degree of neuronal activation, and by quantifying nerve growth factor (NGF) expression in rats. Following caffeine administration for 14 days, a urodynamic study was performed to assess the changes to bladder function. Subsequently, immunohistochemical staining to identify the expression of c­Fos and NGF in the central micturition areas was performed. Ingestion of caffeine increased bladder smooth muscle contraction pressure and time as determined by cystometry. Expression levels of c­Fos and NGF in all central micturition areas were significantly increased following the administration of caffeine. The effects on contraction pressure and time were the most potent and expression levels of c­Fos and NGF were greatest at the lowest dose of caffeine. These results suggest that caffeine facilitates bladder instability through enhancing neuronal activation in the central micturition areas.

Spontaneous voiding by mice reveals strain-specific lower urinary tract function to be a quantitative genetic trait.

Lower urinary tract (LUT) symptoms become prevalent with aging and affect millions; however, therapy is often ineffective because the etiology is unknown. Existing assays of LUT function in animal models are often invasive; however, a noninvasive assay is required to study symptom progression and determine genetic correlates. Here, we present a spontaneous voiding assay that is simple, reproducible, quantitative, and noninvasive. Young female mice from eight inbred mouse strains (129S1/SvImJ, A/J, C57BL/6J, NOD/ShiLtJ, NZO/H1LtJ, CAST/EiJ, PWK/PhJ, and WSB/EiJ) were tested for urination patterns on filter paper. Repeat testing at different times of the day showed minimal within-individual and within-strain variations, but all parameters (spot number, total volume, percent area in primary void, corner voiding, and center voiding) exhibited significant variations between strains. Calculation of the intraclass correlation coefficient, an estimate of broad-sense heritability, for each time of day and for each voiding parameter revealed highly significant heritability [spot number: 61%, percent urine in primary void: 90%, and total volume: 94% (afternoon data)]. Cystometrograms confirmed strong strain-specific urodynamic characteristics. Behavior-voiding correlation analysis showed no correlation with anxiety phenotypes. Diagnostically, the assay revealed LUT symptoms in several systems, including a demonstration of voiding abnormalities in older C57BL/6J mice (18-24 mo), in a model of protamine sulfate-induced urothelial damage and in a model of sucrose-induced diuresis. This assay may be used to derive pathophysiological LUT readouts from mouse models. Voiding characteristics are heritable traits, opening the way for genetic studies of LUT symptoms using outbred mouse populations.

Deletion of Dicer in smooth muscle affects voiding pattern and reduces detrusor contractility and neuroeffector transmission.

MicroRNAs have emerged as important regulators of smooth muscle phenotype and may play important roles in pathogenesis of various smooth muscle related disease states. The aim of this study was to investigate the role of miRNAs for urinary bladder function. We used an inducible and smooth muscle specific Dicer knockout (KO) mouse which resulted in significantly reduced levels of miRNAs, including miR-145, miR-143, miR-22, miR125b-5p and miR-27a, from detrusor preparations without mucosa. Deletion of Dicer resulted in a disturbed micturition pattern in vivo and reduced depolarization-induced pressure development in the isolated detrusor. Furthermore, electrical field stimulation revealed a decreased cholinergic but maintained purinergic component of neurogenic activation in Dicer KO bladder strips. The ultrastructure of detrusor smooth muscle cells was well maintained, and the density of nerve terminals was similar. Western blotting demonstrated reduced contents of calponin and desmin. Smooth muscle α-actin, SM22α and myocardin were unchanged. Activation of strips with exogenous agonists showed that depolarization-induced contraction was preferentially reduced; ATP- and calyculin A-induced contractions were unchanged. Quantitative real time PCR and western blotting demonstrated reduced expression of Cav1.2 (Cacna1c). It is concluded that smooth muscle miRNAs play an important role for detrusor contractility and voiding pattern of unrestrained mice. This is mediated in part via effects on expression of smooth muscle differentiation markers and L-type Ca(2+) channels in the detrusor.

Rodent models for urodynamic investigation.

Rodents, most commonly rats, mice, and guinea pigs are widely used to investigate urinary storage and voiding functions, both in normal animals and in models of disease. An often used methodology is cystometry. Micturitions in rodents and humans differ significantly and this must be considered when cystometry is used to interpret voiding in rodent models. Cystometry in humans requires active participation of the investigated patient (subject), and this can for obvious reasons not be achieved in the animals. Cystometric parameters in rodents are often poorly defined and do not correspond to those used in humans. This means that it is important that the terminology used for description of what is measured should be defined, and that the specific terminology used in human cystometry should be avoided. Available disease models in rodents have limited translational value, but despite many limitations, rodent cystometry may give important information on bladder physiology and pharmacology. The present review discusses the principles of urodynamics in rodents, techniques, and terminology, as well as some commonly used disease models, and their translational value.

Genetic influences are important for most but not all lower urinary tract symptoms: a population-based survey in a cohort of adult Swedish twins.

BACKGROUND: The relative importance of genetic and environmental factors for the occurrence of lower urinary tract symptoms (LUTS) is poorly understood. OBJECTIVE: To (1) estimate the prevalence of urinary incontinence (UI), overactive bladder (OAB), and other LUTS and (2) to assess the heritability of these symptoms. DESIGN, SETTING, AND PARTICIPANTS: Cross-sectional survey of LUTS in a national population-based cohort of Swedish twins 20-46 yr of age (n=42 582) from the Swedish Twin Registry. MEASUREMENTS: Prevalence rates were determined and heritability of LUTS (in female twins) was assessed using indicators of twin similarity. RESULTS AND LIMITATIONS: A total of 25 364 twins completed the questionnaire (response rate: 59.6%). LUTS were more common in women (UI: 7%; OAB: 9%; nocturia: 61%; micturition frequency: 18%) than in men (UI: 1%; OAB: 5%; nocturia: 40%; micturition frequency: 11%), and prevalence increased with age. The strongest genetic effects were observed for UI, frequency, and nocturia. The lowest estimate for genetic effects was observed for OAB where environmental effects dominated, and more specifically shared family environment accounted for a third or more of the total variation. For stress UI, a fifth of the total variation in susceptibility to the disorder could be attributed to shared environment. Nonshared environmental effects were seen in the range of 45-65% for the various LUTS. The prevalence of LUTS was low in the men, and there were too few male cases to compute measures of similarity or heritability estimates. CONCLUSIONS: This study provides robust evidence of a genetic influence for susceptibility to UI, frequency, and nocturia in women. In contrast, shared environmental factors seem more important for the predisposition to develop OAB, which may reflect familial patterns such as learning from parental behaviours.

A new diabetic mouse model derived from the ddY strain.

Selective breeding was used to isolate hyperglycemic (ddY-H) or normoglycemic (ddY-L) mice that had been induced by fasting and refeeding. Serum glucose levels 12 h after 48 h-fasting were high in male ddY-H mice, but relatively low in male ddY-L mice compared with control ddY mice. Glucose tolerance was impaired in ddY-H mice maintained with standard chow pellets ad libitum at 12 weeks of age, and serum glucose and insulin levels were significantly increased after overnight fasting at 15 weeks of age. ddY-L mice indices did not differ from ddY mice indices, suggesting that insulin resistance is spontaneously induced in ddY-H mice. Increases in urinary excretion and urinary sugar accompanied by increased body mass were observed in all ddY-H mice, but not in ddY or ddY-L mice, at 27 weeks of age, indicating the induction of diabetic symptoms. Cross-mating between ddY-H and ddY-L mice was used to certify the genetic involvement in impaired glucose tolerance. This was not induced in mice born from male ddY-H and female ddY-L mice, or from female ddY-H and male ddY-L mice. In conclusion, ddY-H mice are a useful diabetic mouse model that show spontaneously induced insulin resistance followed by diabetic symptoms that are maintained by standard chow pellets. Their characteristics are recessively inherited, and ddY-L mice are an appropriate choice as control mice.

Correlation between pharmacologically-induced changes in cystometric parameters and spinal c-Fos expression in rats.

The inhibition of bladder sensory transmission is critical for the pharmacotherapy of urine storage symptoms. The purpose of this study is to examine the correlation between pharmacologically-induced changes in cystometric parameters and spinal c-Fos expression in anesthetized rats with bladder hyperactivity induced by the intravesical infusion of acetic acid. Animals were intravenously infused with either oxybutynin (OXY), a muscarinic receptor antagonist, tamsulosin (TAM), an alpha1-adrenoceptor antagonist, CL316243 (CL), a beta3-adrenoceptor agonist, or saline. Morphine (MOR) treatment served as a positive control to inhibit bladder afferent activity. Intermicturition intervals, micturition pressure and pressure threshold were measured after intravesical acetic acid infusion. Animals were then perfused and spinal cords were removed. Sections from the L6 spinal cord were immunostained with an anti-c-Fos antibody, and c-Fos positive cells in the dorsal region were counted. CL and MOR significantly increased intermicturition intervals, whereas OXY and TAM had no significant effect on intermicturition intervals. While TAM and MOR did not affect the micturition pressure, OXY and CL caused a significant decrease. Pressure threshold was significantly decreased by CL and increased by MOR. All drugs significantly decreased the number of c-Fos positive cells with the following order of efficacy: MOR>CL>OXT>TAM. The number of c-Fos positive cells in each animal from all treatment groups was negatively correlated with its average intermicturition interval and pressure threshold, but not with its micturition pressure. Bladder afferent activity is suppressed by several clinically proven mechanisms as measured by c-Fos expression, despite the varied effects on cystometric parameters of each pharmacological treatment.

Influence of genetic background and gender on bladder function in the mouse.

Genetically targeted animals are used throughout research to investigate the role genes play in biological function, including the lower urinary tract. Generation of transgenic mice involves backcrossing for successive generations. Parental strain background genes can interact with the mutated gene potentially affecting interpretation of the mutant phenotype. Differences in physiological phenotypes may also be influenced by gender. The present study evaluated bladder function in five strains of male and female mice, 129S6/SvEvTac, A/J, B6129F1/Tac, BALB/cAnNCrL and C57BL/6NTacFBr. Urodynamic parameters were analyzed during infusion of saline (threshold and void volume, non-voiding contractions, pressure threshold and bladder contraction amplitude) in conscious mice and using voluntary urination in freely moving mice placed on filter paper (number of small and large diameter urine spots), which represent commonly used techniques in preclinical characterization of bladder function. Female BALB/c mice exhibited a significantly larger number of non-voiding contractions and urine dripping (increased number of small urine spots) compared to other female mice. Male BALB/c mice did not share this phenotype. Significant differences in threshold and void volumes were also noted amongst strains and genders. The numbers of large diameter urine spots differed amongst female, and not male, mouse strains. Gender differences were observed between sexes of the same strain in both large and small urine spots. These data demonstrate that genetic background and gender can influence bladder function in the mouse. These differences have a significant impact on the choice of strain and gender when investigating the effects of genetic manipulation on the micturition reflex.

Three murine anxiety models: results from multiple inbred strain comparisons.

The literature surrounding rodent models of human anxiety disorders is discrepant concerning which models reflect anxiety-like behavior distinct from general activity and whether different models are measuring the same underlying constructs. This experiment compared the responses of 15 inbred mouse strains (129S1/SvlmJ, A/J, AKR/J, BALB/cByJ, C3H/HeJ, C57BL/6J, C57L/J, CBA/J, CE/J, DBA/2J, FVB/NJ, NZB/B1NJ, PL/J, SJL/J and SWR/J) in three anxiety-like behavioral tasks (light/dark test, elevated zero-maze and open field) to examine whether responses were phenotypically and/or genetically correlated across tasks. Significant strain differences were found for all variables examined. Principal components analyses showed that variables associated with both activity and anxiety-like behaviors loaded onto one factor, while urination and defecation loaded onto another factor. Our findings differ from previous research by suggesting that general activity and anxiety-related behaviors are linked, negatively correlated and cannot easily be dissociated in these assays. However, these findings may not necessarily generalize to other unconditioned anxiety-like behavioral tests.

Effect of an increase in brain serotonin on the osmoregulatory response to a hypo- or hyperosmotic load in Wistar and vasopressin-deficient Brattleboro rats.

Serotonin and its receptor agonists stimulate the release of arginine vasopressin (AVP) into peripheral blood under intraventricular injection. To test the hypothesis that brain serotonin can modulate the development of natural osmoregulatory responses, the effect of an increase in endogenous brain serotonin on the response to an intragastric hypo- or hyperosmotic loading was studied in Wistar and AVP-deficient Brattleboro rats. 5-Hydroxytryptophan (5-HTP), the rate-limiting serotonin biosynthesis precursor known to increase the brain level of serotonin, was injected intraperitoneally (5 mg/100 g body weight). The renal functional parameters (glomerular filtration rate [GFR], free water reabsorption, and urine flow rate) were monitored during the 4 h after intragastric infusion of water or a 2% NaCl solution (5% of body weight). Plasma AVP was measured by radioimmunoassay. In Wistar rats, intraperitoneal injection of 5-HTP at the same time as water loading prevented the development of the renal diuretic response: there was no increase in urine flow rate and GFR, and free water reabsorption remained at the high level. In AVP-deficient Brattleboro rats, unlike Wistar rats, 5-HTP treatment was without effect on the renal function parameters. In Wistar rats, injection of 5-HTP at the peak of water diuresis produced an abrogation of the diuretic response to water loading due to the increase in free water reabsorption. Plasma AVP increased from 1.2 +/- 0.4 to 4.2 +/- 1.6 pg/ml (n = 8 in each group, p < 0.01). Hyperosmotic treatment of Wistar rats with a 2% NaCl solution stimulated AVP secretion compared to baseline (from 3.2 +/- 0.1, n = 7 to 5.6 +/- 0.9, n = 7, p < 0.01), and the saluretic response developed on the background of high free water reabsorption. When injected concomitantly with NaCl solution, 5-HTP revealed no additive effect on plasma AVP and on free water reabsorption. We conclude that the 5-HTP-caused increase in brain serotonin contributed significantly to the dynamics of changes in the osmoregulatory response to the hypo-osmotic challenge due to stimulation of AVP secretion. 5-HTP had no additive effect on the osmoregulatory response to hyperosmotic loading. Peripherally injected 5-HTP had no effect on the renal function, being absent in AVP-deficient Brattleboro rats.

Differential gene expression of cholinergic muscarinic receptor subtypes in male and female normal human urinary bladder.

OBJECTIVES: To study the mRNA expression of each muscarinic receptor subtype in bladder areas involved in micturition, such as the bladder dome, neck, and trigone. Our study focused on the analysis of the gene expression of muscarinic receptors in the human male and female urinary bladder. Other than the well-known role of bladder parasympathetic innervation, an extensive study of the muscarinic receptor mRNA distribution in male and female urinary bladder is still lacking. METHODS: The study was carried out on 5 female (age 56 +/- 10 years) and 5 male (age 70 +/- 9 years) patients. The patients selected for this study did not have any lower urinary tract symptoms, as determined by International Prostate Symptom Score questionnaire. The mRNAs encoding muscarinic receptor subtypes were assessed by reverse transcription-polymerase chain reaction, followed by Southern blot analysis. RESULTS: Using a molecular approach, we demonstrated the presence of all muscarinic receptor subtypes in the different urinary bladder areas involved in micturition; in particular, our data indicated that mRNAs encoding muscarinic receptors are largely expressed in all examined bladder areas, both in men and women, although with some remarkable differences and a peculiar distribution. CONCLUSIONS: Our results indicate that the pharmacology of the human bladder may be more complex than previously recognized. Furthermore, the choice to study each biopsy as a single sample and not use a pool of tissues allowed us to point out the individual variability between subjects and sex-related differences in the expression profile of muscarinic receptor subtype mRNAs.