bladder-smooth-muscle-strip-contractility-as-method-to-evaluate-lower

Bladder Smooth Muscle Strip Contractility as a Method to Evaluate Lower Urinary Tract Pharmacology

We describe an in vitro method to measure bladder smooth muscle contractility, and its use for investigating physiological and pharmacological properties ...

University of Pittsburgh School of Medicine

The ion channel transient receptor potential vanilloid (TRPV) 4 can be activated by hypo-osmolarity, heat, or certain lipid compounds. Here, we demonstrate expression of functional TRPV4 protein in the urothelium lining the renal pelvis, ureters, urinary bladder, and urethra. Exposure of cultured rat urothelial cells from the urinary bladder to the TRPV4-selective agonist 4alpha-phorbol 12,13-didecanoate (4alpha-PDD) promoted Ca2+ influx, evoked ATP release, and augmented the ATP release evoked by hypo-osmolarity. In awake rats during continuous infusion cystometrograms, intravesical administration of 4alpha-PDD (10-100 microM) increased maximal micturition pressure by 51%, specifically by augmenting the portion of each intravesical pressure wave that follows high-frequency urethral oscillations and voiding. This unusual pharmacological effect was prevented by intravesical pretreatment with the nonselective ATP receptor antagonist, pyridoxal phosphate-6-azophenyl-2',4'-disulfonic acid (100 microM), systemic treatment with the selective P2X3 purinergic antagonist 5-([(3-phenoxybenzyl)[1S)-1,2,3,4-tetrahydro-1-naphthalenyl]amino]carbonyl)-1,2,4-benzenetricarboxylic acid (A317491) (250 micromol/kg), or urethane anesthesia, but was unaffected by capsaicin pretreatment (100 mg/kg s.c.) or denervation of the urethral sphincter. 4Alpha-PDD (1-100 microM) did not alter the contractility to electrical stimulation of excised bladder strips. We conclude that activation of urothelial TRPV4 by 4alpha-PDD and release of mediators such as ATP trigger a novel neural mechanism that regulates the late phase of detrusor muscle contraction after micturition. These data raise the possibility that TRPV4 channels in the urothelium could contribute to abnormal bladder activity.

Activation of urothelial transient receptor potential vanilloid 4 by 4alpha-phorbol 12,13-didecanoate contributes to altered bladder reflexes in the rat.

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.

Heterogeneity of muscarinic receptor-mediated Ca2+ responses in cultured urothelial cells from rat.

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 microM) and were absent in rats pretreated with capsaicin to desensitize C-fiber afferent nerves. Low concentrations of OxoM (5 microM) decreased voiding frequency by approximately 30%, an effect blunted by inhibiting nitric oxide (NO) synthesis with L-NAME (N(omega)-nitro-L-arginine methyl ester hydrochloride; 5 mg/kg; i.v.). High concentrations of OxoM (40 microM) increased voiding frequency by approximately 45%, 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 microg/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.

Activation of muscarinic receptors in rat bladder sensory pathways alters reflex bladder activity.

Transient receptor potential (TRP) and acid-sensing ion channels (ASIC) are molecular detectors of chemical, mechanical, thermal, and nociceptive stimuli in sensory neurons. They have been identified in the urothelium, a tissue considered part of bladder sensory pathways, where they might play a role in bladder function. This study investigated functional properties of TRP and ASIC channels in cultured urothelial cells from the rat using patch-clamp and fura 2 Ca(2+) imaging techniques. The TRPV4 agonist 4alpha-phorbol-12,13 didecanoate (4alpha-PDD; 1-5 microM) and the TRPA1/TRPM8 agonist icilin (50-100 microM) elicited transient currents in a high percentage of cells (>70%). 4alpha-PDD responses were suppressed by the TRPV4 antagonist HC-010961 (10 microM). The TRPV1 agonist capsaicin (1-100 microM) and the TRPA1/TRPM8 agonist menthol (5-200 microM) elicited transient currents in a moderate percentage of cells ( approximately 25%). All of these agonists increased intracellular calcium concentration ([Ca(2+)](i)). Most cells responded to more than one TRP agonist (e.g., capsaicin and 4alpha-PDD), indicating coexpression of different TRP channels. In the presence of the TRPV1 antagonist capsazepine (10 microM), changes in pH induced by HCl elicited ionic currents (pH 5.5) and increased [Ca(2+)](i) (pH 6.5) in approximately 50% of cells. Changes in pH using acetic acid (pH 5.5) elicited biphasic-like currents. Responses induced by acid were sensitive to amiloride (10 microM). In summary, urothelial cells express multiple TRP and ASIC channels, whose activation elicits ionic currents and Ca(2+) influx. These "neuron-like" properties might be involved in transmitter release, such as ATP, that can act on afferent nerves or smooth muscle to modulate their responses to different stimuli.

Functional TRP and ASIC-like channels in cultured urothelial cells from the rat.

Nitric oxide (NO), a neurotransmitter in the lower urinary tract, stimulates soluble guanylyl cyclase (sGC) and in turn cGMP-dependent protein kinase G (PKG) to modulate a number of downstream targets. NO donors reduce bladder hyperactivity in some pathological models but do not affect normal bladder activity in the adult rat. In this study, the NO donor S-nitroso-N-acetyl-DL-penicillamine (SNAP; 100 microM) decreased the amplitude and frequency of spontaneous and carbachol-enhanced contractions in neonatal rat bladder strips, which are intrinsically hyperactive. This effect was blocked by inhibition of sGC and mimicked by application of a membrane-permeable cGMP analog (8-bromo-cGMP, 100 microM). Inhibition of PKG prevented or reversed the inhibitory effects of 8-bromo-cGMP. A portion of the SNAP-mediated inhibition was also dependent upon PKG; however, a short-lasting, sGC-dependent inhibitory effect of SNAP was still present after PKG inhibition. Inhibition of NO synthase with L-NAME (100 microM) did not change the amplitude or frequency of contractions. However, inhibition of endogenous phosphodiesterase (PDE)-5 with zaprinast (25 microM) reduced the amplitude and frequency of phasic contractions and increased the magnitude of inhibition produced by maximal concentrations of SNAP, suggesting that endogenous PDEs are constitutively active and regulate cGMP production. These results suggest that the NO-cGMP-PKG pathway may be involved in inhibitory control of the neonatal rat bladder.

Activation of the nitric oxide-cGMP pathway reduces phasic contractions in neonatal rat bladder strips via protein kinase G.

Voiding dysfunctions, including increased voiding frequency, urgency, or incontinence, are prevalent in the postmenopausal population. Beta(3)-adrenergic receptor (beta(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 beta(3)AR agonists to treat bladder hyperactivity in this setting. OVX increased voiding frequency and decreased bladder capacity by approximately 25% in awake rats and induced irregular cystometrograms in urethane-anesthetized rats. Reverse transcription-polymerase chain reaction revealed three betaARs subtypes (beta(1,2,3)) in bladder tissue, and immunostaining indicated beta(3)AR localization in urothelium and detrusor. Receptor expression was not different in OVX and SHAM rats. The beta(3)AR agonist selectivity of BRL37344 [(+/-)-(R(*),R(*))-[4-[2-[[2-(3-chlorophenyl)-2-hydroxyethyl]amino]propyl]phenoxy]acetic acid sodium hydrate], TAK-677 [(3-((2R)-(((2R)-(3-chlorophenyl)-2-hydroxyethyl)amino)propyl)-1H-indol-7-yloxy)acetic acid], and FK175 [acetic acid, 2-[[(8S)-8-[[(2R)-2-(3-chlorophenyl)-2-hydroxyethyl]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 betaARs. Intravenous injection of each of the beta(3)AR agonists (0.1-500 microg/kg) in anesthetized rats decreased voiding frequency, bladder pressure, and amplitude of bladder contractions. In bladder strips, beta(3)AR agonists (10(-12)-10(-4) 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 beta(3)AR expression or the bladder inhibitory effects of beta(3)AR agonists. This suggests that beta(3)AR agonists should prove effective for the treatment of overactive bladder symptoms in the postmenopausal population.

Effects of beta3-adrenergic receptor activation on rat urinary bladder hyperactivity induced by ovariectomy.

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.

Urothelial beta-3 adrenergic receptors in the rat bladder.

During postnatal development large amplitude spontaneous activity of the neonatal rat bladder changes to a low amplitude adult pattern of activity that leads to improved storage function. Previously, we have shown that spontaneous activity in neonatal rat bladder strips is inhibited by activation of the nitric oxide (NO)-cGMP signaling pathway. In the present experiments we determined if this inhibitory pathway is altered during postnatal development or spinal cord injury.

Developmental and spinal cord injury-induced changes in nitric oxide-mediated inhibition in rat urinary bladder.

To determine if the muscarinic agonist, bethanechol, inhibits the non-cholinergic, atropine-resistant (i.e. putatively purinergic) component of naturally occurring (i.e. reflexogenic) bladder contractions in vivo in the rat, as previously described in vitro. Our second aim was to determine if elevation of endogenous acetylcholine (ACh) with distigmine, an acetylcholine esterase (AChE) inhibitor, could also inhibit non-cholinergic component of reflexogenic bladder contractions.

Exogenous activation of muscarinic receptors decreases subsequent non-muscarinic bladder contractions in vivo in the female rat.

This study evaluated the effects of a 5-HT4 agonist, cisapride, on neuronally evoked smooth muscle responses in bladder, urethra and ileum and compared these effects with those of an acetylcholinesterase inhibitor, distigmine. Electrical field stimulation (EFS) was applied to human bladder and ileum smooth muscle strips from human organ transplant donors and to urethral strips from prostatectomy patients, to evoke neuronally mediated smooth muscle responses. EFS induced contractions in bladder and mixed responses, consisting of contractions and relaxations, in urethra and ileum. Relaxations were mediated by nitric oxide while contractions were partially cholinergic (i.e. atropine sensitive). This atropine sensitive component amounted to~95% in bladder and ~75% in ileum, and it was enhanced by distigmine in a concentration dependent manner (0.1-3 μM; ~100-600% increase in bladder and ~50-250% increase in ileum). Cisapride (0.0003-1 μM) also enhanced bladder contractions (~75-100% increase) but had no effect on urethral contractions or relaxations, and modestly enhanced ileum contractions (~10-40% increase). Facilitatory effects of cisapride were reversed by the specific 5-HT4 receptor antagonist, SB-203186 (3 μM), but were resistant to repeated washing in the bladder. These data indicate that 5-HT4 receptor agonists enhanced EFS-induced contractions in bladder and ileum without an effect on urethra and suggest that it may be possible to enhance bladder activity with a dose of cisapride that is at, or below, those producing gastrointestinal (GI) effects. Although distigmine's maximal facilitation of bladder and GI tract function was greater than that of cisapride, at clinically relevant concentrations cisapride showed much greater efficacy.

Effects of the 5-HT4 receptor agonist, cisapride, on neuronally evoked responses in human bladder, urethra, and ileum.

Time- and vehicle-related variability of bladder and urethral rhabdosphincter (URS) activity as well as cardiorespiratory and blood chemistry values were examined in the acetic acid-induced bladder irritation model in α-chloralose-anesthetized female cats. Additionally, bladder and urethra were evaluated histologically using Mason trichrome and toluidine blue staining. Urodynamic, cardiovascular and respiratory parameters were collected during intravesical saline infusion followed by acetic acid (0.5%) to irritate the bladder. One hour after starting acetic acid infusion, a protocol consisting of a cystometrogram, continuous infusion-induced rhythmic voiding contractions, and a 5 min "quiet period" (bladder emptied without infusion) was precisely repeated every 30 minutes. Administration of vehicle (saline i.v.) occurred 15 minutes after starting each of the first 7 cystometrograms and duloxetine (1mg/kg i.v.) after the 8(th). Acetic acid infusion into the bladder increased URS-EMG activity, bladder contraction frequency, and decreased contraction amplitude and capacity, compared to saline. Bladder activity and URS activity stabilized within 1 and 2 hours, respectively. Duloxetine administration significantly decreased bladder contraction frequency and increased URS-EMG activity to levels similar to previous reports. Cardiorespiratory parameters and blood gas levels remained consistent throughout the experiment. The epithelium of the bladder and urethra were greatly damaged and edema and infiltration of neutrophils in the lamina propria of urethra were observed. These data provide an ample evaluation of the health of the animals, stability of voiding function and appropriateness of the model for testing drugs designed to evaluate lower urinary tract as well as cardiovascular and respiratory systems function.

Stability of the acetic acid-induced bladder irritation model in alpha chloralose-anesthetized female cats.

Bombesin receptors (BB receptors) and/or bombesin related peptides are expressed in the lower urinary tract, though their function and distribution in different species is largely unknown. This study examines whether BB receptor agonists can contract bladder smooth muscle in rats, mice, pigs and humans.

Functional bombesin receptors in urinary tract of rats and human but not of pigs and mice, an in vitro study.

Bombesin receptors (BB receptors) and bombesin related peptides are expressed in the lower urinary tract of rodents. Here we investigated whether in vivo activation of BB receptors can contract the urinary bladder and facilitate micturition in sham rats and in a diabetic rat model of voiding dysfunction.

Excitatory effects of bombesin receptors in urinary tract of normal and diabetic rats in vivo.

In vivo experiments in a diabetic rat model revealed compromised nitrergic urethral relaxations and increased sensitivity to adrenergic agonists. This study evaluated contractile and relaxation properties of urethral smooth muscle after streptozotocin (STZ)-induced diabetes, in vitro, with the aim of determining whether in vivo deficiencies are related to smooth muscle dysfunction. Urethral tissue was collected from adult female Sprague-Dawley rats naive, STZ-treated, vehicle-treated and sucrose-fed at 9-12 week post treatment. Strips from proximal, mid, and distal urethra were placed in tissue baths and stimulated using electric field stimulation (EFS) and pharmacological agents. nNOS staining was evaluated using immunohistochemistry. Phenylephrine (PE, 10μM) contracted all urethral strips with the highest amplitude in mid urethra, in all treatment groups. Likewise, EFS-induced relaxation amplitudes were larger and were observed more frequently in mid urethra. Relaxations were inhibited by the NOS inhibitor, L-NAME (1-100μM). Sodium nitroprusside (0.01-1μM), an NO donor, reversed PE-induced contractions. No statistical differences were observed between treatment groups with respect to any parameters. Qualitative immunohistochemistry showed no differences in the urethral nNOS innervation patterns across the treatment groups. In summary, nitrergic relaxations and adrenergic-induced contractions in the isolated diabetic rat urethra display similar properties to controls, suggesting no dysfunction on the nitrergic or alpha1 adrenergic receptor function in the smooth muscle. This further implies that compromised urethral relaxation and increased adrenergic agonist sensitivity observed in vivo in this model may be due to the disruption of neural signaling between the urethra and the spinal cord, or within the CNS.

Article	Year
Activation of urothelial transient receptor potential vanilloid 4 by 4alpha-phorbol 12,13-didecanoate contributes to altered bladder reflexes in the rat. The Journal of pharmacology and experimental therapeutics\| PubMed ID: 17636010	2007
Heterogeneity of muscarinic receptor-mediated Ca2+ responses in cultured urothelial cells from rat. American journal of physiology. Renal physiology\| PubMed ID: 18272602	2008
Activation of muscarinic receptors in rat bladder sensory pathways alters reflex bladder activity. The Journal of neuroscience : the official journal of the Society for Neuroscience\| PubMed ID: 18287514	2008
Functional TRP and ASIC-like channels in cultured urothelial cells from the rat. American journal of physiology. Renal physiology\| PubMed ID: 19158342	2009
Activation of the nitric oxide-cGMP pathway reduces phasic contractions in neonatal rat bladder strips via protein kinase G. American journal of physiology. Renal physiology\| PubMed ID: 19493964	2009
Effects of beta3-adrenergic receptor activation on rat urinary bladder hyperactivity induced by ovariectomy. The Journal of pharmacology and experimental therapeutics\| PubMed ID: 19515967	2009
Urothelial beta-3 adrenergic receptors in the rat bladder. Neurourology and urodynamics\| PubMed ID: 21046653	2011
Developmental and spinal cord injury-induced changes in nitric oxide-mediated inhibition in rat urinary bladder. Neurourology and urodynamics\| PubMed ID: 21717503	2011
Exogenous activation of muscarinic receptors decreases subsequent non-muscarinic bladder contractions in vivo in the female rat. Life sciences\| PubMed ID: 23399698	2013
Effects of the 5-HT4 receptor agonist, cisapride, on neuronally evoked responses in human bladder, urethra, and ileum. Autonomic neuroscience : basic & clinical\| PubMed ID: 23511063	2013
Stability of the acetic acid-induced bladder irritation model in alpha chloralose-anesthetized female cats. PloS one\| PubMed ID: 24040064	2013
Functional bombesin receptors in urinary tract of rats and human but not of pigs and mice, an in vitro study. Neuropeptides\| PubMed ID: 24054546	2013
Excitatory effects of bombesin receptors in urinary tract of normal and diabetic rats in vivo. Life sciences\| PubMed ID: 24496039	2014
Nitrergic relaxations and phenylephrine contractions are not compromised in isolated urethra in a rat model of diabetes. Autonomic neuroscience : basic & clinical\| PubMed ID: 24656892	2014

F. Aura Kullmann

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