Antimuscarinic drugs affect bladder sensory symptoms such as urgency and frequency, presumably by acting on muscarinic acetylcholine receptors (mAChRs) located in bladder sensory pathways including primary afferent nerves and urothelium. However, the expression and the function of these receptors are not well understood. This study investigated the role of mAChRs in bladder sensory pathways in vivo in urethane anesthetized rats. Intravesical administration of the mAChR agonist oxotremorine methiodide (OxoM) elicited concentration-dependent excitatory and inhibitory effects on the frequency of voiding. These effects were blocked by intravesical administration of the mAChR antagonist atropine methyl nitrate (5 M) and were absent in rats pretreated with capsaicin to desensitize C-fiber afferent nerves. Low concentrations of OxoM (5 M) decreased voiding frequency by ϳ30%, an effect blunted by inhibiting nitric oxide (NO) synthesis with L-NAME (N -nitro-L-arginine methyl ester hydrochloride; 5 mg/kg; i.v.). High concentrations of OxoM (40 M) increased voiding frequency by ϳ45%, an effect blunted by blocking purinergic receptors with PPADS (0.1-1 mM; intravesically). mAChR agonists stimulated release of ATP from cultured urothelial cells. Intravenous administration of OxoM (0.01-5 g/kg) did not mimic the intravesical effects on voiding frequency. These results suggest that activation of mAChRs located near the luminal surface of the bladder affects voiding functions via mechanisms involving ATP and NO release presumably from the urothelium, that in turn could act on bladder C-fiber afferent nerves to alter their firing properties. These findings suggest that the urothelial-afferent nerve interactions can influence reflex voiding function.

Muscarinic receptors (mAChRs) have been identified in the urothelium, a tissue that may be involved in bladder sensory mechanisms. This study investigates the expression and function of mAChRs using cultured urothelial cells from the rat. RT-PCR established the expression of all five mAChR subtypes. Muscarinic agonists acetylcholine (ACh; 10 microM), muscarine (Musc; 20 microM), and oxotremorine methiodide (OxoM; 0.001-20 microM) elicited transient repeatable increases in the intracellular calcium concentration ([Ca(2+)](i)) in approximately 50% of cells. These effects were blocked by the mAChR antagonist atropine methyl nitrate (10 microM). The sources of [Ca(2+)](i) changes included influx from external milieu in 63% of cells and influx from external milieu plus release from internal stores in 27% of cells. The use of specific agonists and antagonists (10 microM M(1) agonist McN-A-343; 10 microM M(2), M(3) antagonists AF-DX 116, 4-DAMP) revealed that M(1), M(2), M(3) subtypes were involved in [Ca(2+)](i) changes. The PLC inhibitor U-73122 (10 microM) abolished OxoM-elicited Ca(2+) responses in the presence of the M(2) antagonist AF-DX 116, suggesting that M(1), M(3), or M(5) mediates [Ca(2+)](i) increases via PLC pathway. ACh (0.1 microM), Musc (10 microM), oxotremorine sesquifumarate (20 microM), and McN-A-343 (1 muM) acting on M(1), M(2), and M(3) mAChR subtypes stimulated ATP release from cultured urothelial cells. In summary, cultured urothelial cells express functional M(1), M(2), and M(3) mAChR subtypes whose activation results in ATP release, possibly through mechanisms involving [Ca(2+)](i) changes.

Voiding dysfunctions, including increased voiding frequency, urgency, or incontinence, are prevalent in the postmenopausal population. ␤ 3 -Adrenergic receptor (␤ 3 AR) agonists, which relax bladder smooth muscle, are being developed to treat these conditions. We utilized the rat ovariectomy (OVX) model to investigate the effect of ovarian hormone depletion on bladder function and the potential for ␤ 3 AR agonists to treat bladder hyperactivity in this setting. OVX increased voiding frequency and decreased bladder capacity by ϳ25% in awake rats and induced irregular cystometrograms in urethane-anesthetized rats. Reverse transcription-polymerase chain reaction revealed three ␤ARs subtypes (␤ 1,2,3 ) in bladder tissue, and immunostaining indicated ␤ 3 AR localization in urothelium and detrusor. Receptor expression was not different in OVX and SHAM rats. ]amino]-6,7,8,9-tetrahydro-5H-benzocyclohepten-2-yl]oxy], ethyl ester, hydrochloride] was confirmed by examining the relative potency for elevation of cAMP in CHOK1 cells overexpressing the various rat ␤ARs. Intravenous injection of each of the ␤ 3 AR agonists (0.1-500 g/kg) in anesthetized rats decreased voiding frequency, bladder pressure, and amplitude of bladder contractions. In bladder strips, ␤ 3 AR agonists (10 Ϫ12-10 Ϫ4 M) decreased baseline tone and reduced spontaneous contractions. BRL37344 (5 mg/kg) and TAK-677 (5 mg/kg) injected intraperitoneally in awake rats decreased voiding frequency by 40 to 70%. These effects were not altered by OVX. The results indicate that OVX-induced bladder dysfunction, including decreased bladder capacity and increased voiding frequency, is not associated with changes in ␤ 3 AR expression or the bladder inhibitory effects of ␤ 3 AR agonists. This suggests that ␤ 3 AR agonists should prove effective for the treatment of overactive bladder symptoms in the postmenopausal population.Lower urinary tract (LUT) dysfunctions, including increased voiding frequency, urgency, incontinence and nocturia, increase in the elderly population and following menopause (Stewart et al., 2003). These dysfunctions could result from hormonally induced changes in bladder contractile and/or relaxing mechanisms. Bladder contractions are triggered by parasympathetic nerves, which release ACh that in turn activates postjunctional muscarinic receptors (mAChRs) in the detrusor. Bladder relaxation is induced by release of norepinephrine from sympathetic nerves, which activates ␤-adrenergic receptors (␤AR) (Fowler et al., 2008

Nitro-oleic acid (OA-NO 2 ), an electrophilic fatty acid by-product of nitric oxide and nitrite reactions, is present in normal and inflamed mammalian tissues at up to micromolar concentrations and exhibits anti-inflammatory signaling actions. The effects of OA-NO 2 on cultured dorsal root ganglion (DRG) neurons were examined using fura-2 Ca 2ϩ imaging and patch clamping. OA-NO 2 (3.5-35 M) elicited Ca 2ϩ transients in 20 to 40% of DRG neurons, the majority (60 -80%) of which also responded to allyl isothiocyanate (AITC; 1-50 M), a TRPA1 agonist, and to capsaicin (CAPS; 0.5 M), a TRPV1 agonist. The OA-NO 2 -evoked Ca 2ϩ transients were reduced by the TRPA1 antagonist 2-(1,3-dimethyl-2,6-dioxo-1,2,3,6-tetrahydro-7H-purin-7-yl)-N-(4-isopropylphenyl) acetamide (HC-030031; 5-50 M) and the TRPV1 antagonist capsazepine (10 M). Patchclamp recording revealed that OA-NO 2 depolarized and induced inward currents in 62% of neurons. The effects of OA-NO 2 were elicited by concentrations Ն5 nM and were blocked by 10 mM dithiothreitol. Concentrations of OA-NO 2 Ն5 nM reduced action potential (AP) overshoot, increased AP duration, inhibited firing induced by depolarizing current pulses, and inhibited Na ϩ currents. The effects of OA-NO 2 were not prevented or reversed by the NO-scavenger carboxy-2-phenyl-4,4,5,5-tetramethylimidazolineoxyl-1-oxyl-3-oxide. A large percentage (46 -57%) of OA-NO 2 -responsive neurons also responded to CAPS (0.5 M) or AITC (0.5 M). OA-NO 2 currents were reduced by TRPV1 (diarylpiperazine; 5 M) or TRPA1 (HC-030031; 5 M) antagonists. These data reveal that endogenous OA-NO 2 generated at sites of inflammation may initially activate transient receptor potential channels on nociceptive afferent nerves, contributing to the initiation of afferent nerve activity, and later suppresses afferent firing.

Aims-To investigate the distribution of beta-3 adrenergic receptors (β 3 ARs) in the rat bladder and to examine the contribution of urothelial β 3 ARs to agonist-induced suppression of bladder reflexes and relaxation of smooth muscle.Methods-Bladder tissue was collected from 8-10 months old female SD rats. In some samples, the urothelium was surgically separated from the smooth muscle. The expression and localization of βAR mRNA and β 3 AR protein were determined using RT-PCR and immmunohistochemistry. Contractile responses to the specific β 3 AR agonists TAK-677 and BRL37344 were measured in bladder strips with or without the urothelium. The contribution of urothelial β 3 AR to the micturition reflex was assessed in continuous cystometry in urethane anesthetized rats using intravesical delivery of β 3 AR agonists.Results-RT-PCR detected mRNA of all βARs in urothelium and smooth muscle. Immunostaining detected β 3 ARs throughout the urothelium, in the smooth muscle, myofibroblastlike cells, and in the peripheral nerves. Ovariectomy did not change the distribution of β 3 ARs in any bladder structure. Intravesical administration of TAK-677 and BRL37344 (1 -5×10 -4 M) decreased voiding frequency and amplitude of bladder contractions. In bladder strips in vitro both β 3 AR agonists (10 -12 -10 -4 M) relaxed the smooth muscle in a concentration-dependent manner to the same extent in strips with and without the urothelium.Conclusions-In addition to their presence in bladder smooth muscle, β 3 ARs are present in the urothelium where their activation may alter reflex voiding via release of factor(s) that act on nonmyocyte structures including the afferent and/or efferent nerves to influence bladder contractility.