Vascular Pharmacology 60 (2014) 84–94
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Neurohumoral regulation of spontaneous constrictions in suburothelial venules of the rat urinary bladder Yuki Shimizu, Satoshi Mochizuki, Retsu Mitsui, Hikaru Hashitani ⁎ Department of Cell Physiology, Nagoya City University Graduate School of Medical Sciences, Nagoya 467-8601, Japan
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Article history: Received 18 October 2013 Received in revised form 30 December 2013 Accepted 3 January 2014 Keywords: Microcirculation Bladder Suburothelium Vasomotion Adrenergic nerve
a b s t r a c t Venules of the bladder suburothelium develop spontaneous phasic constrictions that may play a critical role in maintaining venular drainage of tissue metabolites. We aimed to investigate neurohumoral regulation of the spontaneous venular constrictions (SVCs). Changes in venular diameter of the rat bladder suburothelium were monitored using a video tracking system, whilst the effects of electrical field stimulation (EFS) and bathapplied bioactive substances were investigated. The innervation of the suburothelial microvasculature was examined by immunohistochemistry. EFS (10 Hz for 30 s) induced an increase in the frequency of SVCs that was prevented by phentolamine (1 μM). In phentolamine-pretreated venules, EFS suppressed SVCs with a venular dilatation in a manner attenuated by propranolol (1 μM) or L-nitro arginine (LNA, 10 μM). BRL37344 (1 μM), a β3 adrenoceptor agonist, dilated venules and reduced the frequency of SVCs in an LNA-sensitive manner. ACh (1–10 μM) increased the frequency of SVCs. ATP (1 μM) transiently constricted venules and then caused LNA-sensitive cessation of SVCs associated with a dilatation. Substance P (100 nM) caused a venular constriction, whilst calcitonin gene related peptide (CGRP, 100 nM) caused a dilatation of venules and suppression of SVCs that were not inhibited by LNA. Immunohistochemical staining demonstrated sympathetic as well as substance P- and CGRP-containing nerves running along the venules. Spontaneous constrictions of suburothelial venules are accelerated by sympathetic α-adrenergic stimulation, but suppressed upon β-adrenergic stimulation. In addition, suburothelial venular constrictions appear to be modulated by several bioactive substances that could be released from urothelium or suburothelial sensory nerves. © 2014 Elsevier Inc. All rights reserved.
1. Introduction Overactive bladder (OAB) is a highly prevalent disorder that increases with age in both sexes. Its cause or etiology appears to be complex and multifactorial [27]. Considering the current diagnostic criteria of over active bladder, i.e., urinary urgency, enhanced signal transmission from urothelium to afferent nerves may be a primary cause of overactive bladder symptoms [35]. Normal bladder filling sensation is mediated by stretch-dependent release of ATP from urothelium that acts upon P2X3 purinoceptors located on sensory nerve endings [10,30]. ATP also acts on suburothelial interstitial cells [32] or detrusor smooth muscle cells that may modulate urothelium-afferent nerve signal transmission. Thus excessive releases of ATP could result in urinary urgency. Additionally, other bioactive substances released from the urothelium, e.g., acetylcholine [33] and nitric oxide [5], or from sensory nerves [21], e.g., substance P and CGRP, may also modulate afferent nerve activity.
Abbreviations: LNA, N-nitro-L-arginine; SIN-1, 3-(4-Morpholinyl)sydnonimine; CGRP, calcitonin gene-related peptide; EFS, electrical filed stimulation; SVC, spontaneous venular constriction; OAB, overactive bladder. ⁎ Corresponding author. Tel.: +81 52 8538131; fax: +81 52 8421538. E-mail address: [email protected] (H. Hashitani). 1537-1891/$ – see front matter © 2014 Elsevier Inc. All rights reserved. http://dx.doi.org/10.1016/j.vph.2014.01.002
The suburothelial microcirculation is critical in maintaining bladder cell function. In the normal bladder, blood flow is well maintained during the filling phase, and only transiently decreases during voiding [14]. This maintenance primarily relies on the high compliance properties of the bladder wall that allow low pressure storage of urine. However the intrinsic properties of the microvasculature including their winding arrangement and spontaneous venular constrictions (SVCs) [15,16] may also be beneficial in maintaining bladder microcirculation during storage phase. It has been proposed that overactive bladder results from ischemia and/or reperfusion [9] associated with bladder outlet obstruction, aging or atherosclerosis [2,3,34]. Indeed, alterations in urothelial mediated regulation in ischemic bladders result in detrusor instability [2–4]. Previous studies focused on the arteries/arterioles in terms of their function in supplying oxygen and nutrients to the bladder, whilst the role of venules in regulating the bladder microcirculation has garnered less attention. Of studies that examine bladder venules, it is suggested that diminished venular drainage can result in tissue metabolite accumulation and contribute to ‘urinary urgency’ upon acidification-induced stimulation of TRP channels at sensory nerve endings [8,20]. Thus it is reasonable to assume that improvement of bladder storage symptoms with α-adrenoceptor antagonists [22] or PDE5 inhibitors [25] may be attributed to their vasodilator action on the suburothelial microvasculature.
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β3-Adrenoceptor agonists may also exert an influence by improving the microcirculation within the bladder wall [1]. For example, mirabegron, a β3-adrenoceptor agonist, reduces bladder hyperactivity in a rat model of chronic bladder ischemia [28]. The suburothelial microvasculature runs within the connective tissue layer just beneath the urothelium, where both efferent and afferent nerves are abundant, suggesting their function could readily be affected by neurohumoral substances released from nerves as well as the urothelium. We aimed to investigate the neurohumoral regulation of SVCs in the rat bladder since any imbalance between excitatory and inhibitory vasoactive substances may result in suburothelial microcirculation dysfunction. Agents that improve the microcirculation are potential tools for the treatment or even prophylactic treatment of overactive bladder. 2. Materials and methods
Specimens were incubated with PBS containing 0.3% Triton X-100 for 10 min, incubated with Block Ace (Dainippon Seiyaku) for 20 min and incubated with primary antibodies diluted in PBS containing 2% bovine serum albumin and 0.3% Triton X-100 for 4 days at 4 °C. Primary antibodies used in the present study were as follows: mouse monoclonal antibody for α smooth muscle actin (αSMA; 1:500, clone 1A4, Sigma), rabbit anti tyrosine hydroxylase (TH) antibody (1:1000, Millipore), rabbit anti-SP antibody (1:1000, Immunostar) and rabbit anti-CGRP antibody (1:50, Progen Biotechnik). Specimens were then incubated with biotinylated swine anti-rabbit IgG antibody (1:300, Dako) for 30 min at room temperature. Specimens were incubated with Alexa 488-conjugated streptavidin (10 μg/ml, Jackson ImmunoResearch) and Cy-3-conjugated goat anti-mouse IgG antibody (2.5 μg/ml, Millipore) for 2 h at room temperature and coverslipped with a fluorescence mounting medium (Dako). Whole mounts were examined using a confocal laser scanning microscope (LSM 5 PASCAL, Zeiss).
2.1. Animal and ethical approval
2.6. Solutions and drugs
Male Wistar rats 6 to 8 weeks old were used throughout this study. They were housed, cared for and acclimatized (before the experiments). For experiments, rats were anesthetized and exsanguinated by decapitation according to procedures approved by the Nagoya City University Medical School Experimental Animal Committee.
The composition of PSS was (in mM): NaCl, 120; KCl, 4.7; MgCl2, 1.2; CaCl2, 2.5; NaHCO3, 15.5; KH2PO4, 1.2 and glucose, 11.5. The pH of PSS was 7.2 when bubbled with 95% O2 and 5% CO2, and the measured pH of the recording bath was approximately 7.4. The drugs used were acetylcholine (ACh), ADP, ATP, αβ-methylene ATP (αβ-MeATP), BaCl2, BRL37344, human CGRP, phentolamine, propranolol, N-ω-nitro-L-arginine (LNA), 3-(4-morpholinyl)sydnonimine (SIN-1), substance P and tetrodotoxin. All drugs were dissolved in distilled water except SIN-1 which was dissolved in ethanol. The final concentration of solvents in PSS did not exceed 1:1000.
2.2. Tissue preparation The bladder was removed, cut open and pinned in a dissecting dish with the urothelial side uppermost. The mucosal layer was dissected away from the detrusor smooth muscle layer, and the urothelium was carefully removed using ophthalmology scissors leaving the suburothelial layer. 2.3. Video imaging Suburothelial preparations were superfused with warmed (36 °C) physiological salt solution (PSS) at a constant flow rate (about 2 ml/ min). Venules were readily distinguished from arterioles by their larger diameters and thinner walls as well as their regularly occurring SVCs. Changes in the diameter of venules were recorded with a video camera, and analyzed using Diamtrak, video edge detection software. Electrical field stimulation (EFS) was applied to preparations by passing brief currents (10 Hz, pulse width 50 μs) between a pair of platinum electrodes in the recording chamber. The neural selectivity of evoked responses was confirmed by their sensitivity to tetrodotoxin (1 μM). 2.4. Intracellular calcium imaging Suburothelial preparations were incubated in low Ca 2 + PSS ([Ca2 +] o = 0.1 mM) containing 1 μM fluo-8 AM (AAT Bioquest, Sunnyvale, CA, USA) and cremphor EL (0.01%, Sigma) for 30 min at 35 °C. Following incubation, preparations were superfused with dye-free PSS and illuminated at 495 nm with fluorescence emissions (above 515 nm) captured as described previously [10,11]. Relative amplitudes of Ca2+ transients were expressed as ΔFt/F0 = (Ft − F0) / F0, where Ft is the fluorescence generated by an event, and baseline F0 is the basal fluorescence. 2.5. Immunohistochemistry Suburothelial whole mount preparations were immersed in fixative containing 2% formaldehyde and 15% saturated picric acid in 0.1 M phosphate buffer (pH 7.4) for 10 min, and then further immersed in the same fixative overnight at 4 °C. The fixed specimens were washed in dimethyl sulfoxide (DMSO) and then in PBS.
2.7. Calculations and statistics Measured values are shown as the mean ± SD. Statistical significance was tested using the paired t test with P b 0.05 considered significant. The number of tissues is denoted ‘n’, whilst the number of animals is denoted ‘N’. Peak amplitude of SVCs was measured as the value from the basal diameter to the peak of constrictions. Frequency was calculated as an average during 3 min either prior to (control) or during drug application, or was calculated during EFS. 3. Results 3.1. General observations Consistent with our previous report [15], suburothelial venules developed spontaneous phasic constrictions. In 54 suburothelial venules (n = 54, N = 48), SVCs were associated with a transient reduction in their diameter to 78.5 ± 13.2% of their resting diameter (70.9 ± 24.9 μm) and occurred at a frequency of 4.7 ± 1.7 min− 1. During preliminary experiments, a substantial delay between EFS and the onset of venular constrictions were evident. Therefore, the time course of nerve-evoked responses in venules and arterioles was compared by visualizing their Ca2+ transients. EFS (10 Hz for 1 s) triggered a prompt Ca2+ transient in nerve fibers that was immediately followed by Ca2+ transients in the arteriolar smooth muscle cells with a delay of 0.57 ± 0.04 s (n = 5, N = 4, Fig. 1A, B). Subsequently, EFSinduced Ca2+ transients in venular smooth muscle cells were observed with a delay of 2.1 ± 0.17 s (n = 5, N = 4, Fig. 1A, B, Supplement movie 1). Suburothelial venules were immunoreactive for α smooth muscle actin (αSMA, Fig. 1C a, b, d), whilst arterioles showed relatively weak αSMA immunoreactivity (Fig. 1Ca, b). Both arterioles (Fig. 1Cb, c) and venules (Fig. 1Cb, c) were innervated, and varicose sympathetic nerve fibers immunopositive for tyrosine hydroxylase (TH) run along the microvasculature as well as transversely over venules (Fig. 1Cd, e).
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3.2. Effects of EFS on SVCs In suburothelial venules, EFS (10 Hz for 30 s) induced an increase in the frequency of SVCs (from 4.7 ± 1.4 min− 1 to 11.3 ± 2.4 min− 1,
P b 0.05) associated with a sustained reduction in their diameter (76.9 ± 13.7% of control diameter, n = 15, N = 15, P b 0.05, Fig. 2A). These excitatory effects were prevented by phentolamine (1 μM, n = 12, N = 12), such that subsequent EFS induced a venular dilatation
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Fig. 2. Neural regulation of spontaneous venular constrictions. In a suburothelial venule, EFS (under bar, 10 Hz for 30 s) increased the frequency of spontaneous constrictions and caused a sustained constriction (A). In the same venule that had been treated with phentolamine (10 μM) for 20 min, EFS reduced the frequency of spontaneous constrictions with a small dilatation (B). Subsequent propranolol prevented the inhibitory effects of EFS (C). Dotted lines indicate basal diameter. Effects of EFS and phentolamine on the venular diameter and the frequency of spontaneous constrictions were summarized in (C) and (D), respectively.
(112.2 ± 4.2% of control diameter, n = 5, N = 5, P b 0.05, Fig. 2B, D) with a reduction in the frequency of SVCs (from 5.4 ± 1.4 min−1 to 2.6 ± 0.4 min−1, P b 0.05, Fig. 2B, E) that was prevented by propranolol (1 μM, n = 5, N = 5, Fig. 2C). In phentolamine-pretreated venules, EFS-induced venular dilatation was accompanied with either a cessation of SVCs (n = 4, N = 4, Fig. 3A) or a reduction in their frequency (from 5.2 ± 1.1 min− 1 to 2.4 ± 0.7 min−1, n = 10, N = 10, P b 0.05). LNA (10 μM) increased the frequency of SVCs (from 5.4 ± 0.9 min− 1 to 8.1 ± 1.5 min−1, n = 7, N = 7, P b 0.05), and caused a sustained reduction in the basal
diameter (81.9 ± 7.5% of control diameter, n = 7, N = 7, P b 0.05) associated with a suppression of SVC amplitude. In preparations which had been treated with LNA for 30 min, EFS failed to induce venular dilatation (Fig. 3B, C, D). BRL37344 (1 μM), a β3-adrenoceptor agonist, reduced the frequency of SVCs (from 4.7 ± 0.6 min − 1 to 2.5 ± 0.6 min − 1 , n = 6, N = 6, P b 0.05, Fig. 4A) with a venular dilatation (125.7 ± 13.6% of control diameter, P b 0.05). In LNA-pretreated preparations, BRL37344 failed to suppress SVCs (n = 5, N = 5, Fig. 4B, C, D).
Fig. 1. Sympathetic innervation to venules of the bladder suburothelium. (A) Sequential fluo-8 fluorescence Ca2+ images with a frame interval of 770 ms showing EFS (10 Hz for 1 s)induced Ca2+ transient in a nerve fiber (ROI 1), arteriolar smooth muscle (ROI 2) and venular smooth muscle (ROI 3) in a bladder suburothelial preparation. (B) EFS (under bar, 10 Hz for 1 s) evoked a Ca2+ transient in a nerve fiber (black) that was immediately followed by a Ca2+ transient in an arteriole (magenta), and subsequently induced a Ca2+ transient in a venule (blue). (C) Immunoreactivity for α-smooth muscle actin (αSMA) revealed a suburothelial arteriole (A) and venule (V) in the rat bladder (a). Varicose sympathetic nerve fibers immunopositive for tyrosine hydroxylase (TH) innervated both the arteriole and venule (b, c). Immunoreactivity for αSMA in the venule is brighter than that of the arteriole. Several varicose sympathetic fibers projecting to suburothelial venule (V) were detected in another specimen (d, e). Scale bars indicate 50 μm (a) and 20 μm (d), respectively.
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Fig. 3. Effect of LNA on β-adrenergic relaxation. In a suburothelial venule that had been treated with phentolamine (10 μM), EFS (under bar, 10 Hz for 30 s) evoked a venular dilatation with a cessation of spontaneous constrictions (A). In the same venule that had been treated with LNA (10 μM) for 30 min. EFS-induced inhibition of spontaneous constrictions was largely attenuated (B). Dotted lines indicate basal diameter. Effects of EFS and LNA on the venular diameter and the frequency of spontaneous constrictions were summarized in (C) and (D), respectively.
3.3. Effect of ACh and SIN-1 on SVCs Since urothelium-derived ACh may affect suburothelial microvasculature function, the effects of bath-applied ACh on venular contractility were examined. Unexpectedly, ACh (1 μM) increased the frequency of SVCs (from 4.5 ± 1.8 min−1 to 5.8 ± 2.1 min−1, P b 0.05) and caused a reduction in the basal diameter of the venules (89.2 ± 12.4% of control diameter, n = 14, N = 10, P b 0.05, Fig. 5A). A higher concentration of ACh (10 μM) further increased the frequency of SVCs to 6.6 ± 1.9 min− 1, and reduced venular diameter (83.7 ± 15.1% of control diameter, Fig. 5A). Vasorelaxant action of nitric oxide (NO), a predominant endotheliumderived relaxing factor, in suburothelial venules, was evaluated using SIN-1, a NO donor. SIN-1 (1 μM) caused a marked venular dilatation (117 ± 12.7% of control diameter, n = 9, N = 9, P b 0.05), and either reduced the frequency of SVCs (from 3.7 ± 0.5 min− 1 to 2.4 ± 0.75 min−1, n = 3, N = 3, P b 0.05, Fig. 5B) or prevented their generation (n = 6, N = 6). 3.4. Comparative effects of ACh on arteriolar and venular contractility One might argue that the lack of ACh-induced dilatation of the venules may be attributed to damages to their endothelium. Thus effects of ACh on suburothelial arterioles running along the venules were investigated. In suburothelial arterioles, Ba2+ (500 μM), a blocker for inward rectifier potassium channels [23], caused a sustained reduction in the basal diameter (61.9 ± 7.2% of control diameter, n = 10, N = 9, P b 0.05). Subsequent ACh (10 μM) caused a dilatation of the Ba2 +-contracted
arterioles to 97.8 ± 11.7% of control diameter. ACh-induced dilatations were partially reversed by LNA (10 μM, 68.2 ± 17.2% of control diameter, n = 6, N = 6, Fig. 6A, C), suggesting that ACh was capable of releasing NO from the arteriolar endothelium. In suburothelial venules, Ba2+ (500 μM), increased the frequency of SVCs (from 3.5 ± 1.1 min− 1 to 5.4 ± 2.3 min− 1, n = 7, N = 7, P b 0.05) and often induced long lasting constrictions which appeared to be composed of fused SVCs with a reduction in the basal diameter (94.0 ± 4.2% of control diameter, P b 0.05, Fig. 6B). ACh (10 μM) caused a sustained constriction (82.8 ± 14.8% of control diameter, n = 10, N = 10, P b 0.05, Fig. 6B, D). During the sustained constrictions, individual SVCs were fused and undistinguishable (n = 8, N = 8) or their frequency was further increased (6.3 ± 0.6 min−1, n = 2, N = 2). 3.5. Effects of ATP on SVCs ATP (1 μM), a major urothelial transmitter, transiently increased the frequency of SVCs (from 4.9 ± 0.7 min− 1 to 11.5 ± 2.3 min−1, P b 0.05) with a reduction in the basal diameter (78.5 ± 9.2% of control diameter, P b 0.05), and then prevented SVCs with a dilatation of venular diameter (123.6 ± 15.1% of control diameter, n = 8, N = 8, P b 0.05, Fig. 7A). In LNA-pre-treated venules, ATP caused a small constriction (88.9 ± 5.4% of control diameter, n = 5, N = 5, P b 0.05, Fig. 7B). Upon withdrawal of ATP, there was a small dilatation (105 ± 2.2% of control diameter, P b 0.05) and reduction in the frequency of SVCs (from 10.4 ± 2.5 min−1 to 8.9 ± 2.1 min−1, P b 0.05). ADP (1 μM), a P2Y agonist, caused a transient increase in SVCs (from 4.9 ± 0.9 min− 1 to 10.1 ± 1.1 min− 1, P b 0.05) with a reduction in venular diameter (80.2 ± 6.6% of control diameter, P b 0.05),
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Fig. 4. Effect of BRL 37344 on venular spontaneous constrictions. In a suburothelial venule, BRL 37344 (1 μM) caused a dilatation associated with a reduction in the frequency of spontaneous constrictions (A). In the same venule that had been treated with LNA (10 μM) for 30 min. BRL 37344 (1 μM) failed to suppress spontaneous constrictions (B). Dotted lines indicate basal diameter. Effects of BRL 37344 and LNA on the venular diameter and the frequency of spontaneous constrictions were summarized in (C) and (D), respectively.
and then caused a cessation of SVCs with a dilatation of venules (121.8 ± 7.5% of control diameter, n = 5, N = 5, P b 0.05, Fig. 7C). In LNA-pretreated venules, ADP caused a small constriction (92.6 ± 6.9% of control diameter, n = 3, N = 3, P b 0.05, Fig. 7D). After withdrawal of ADP, a small dilatation (105.2 ± 1.1% of control diameter, P b 0.05)
and reduction in the frequency of SVCs (from 9.7 ± 0.6 min− 1 to 7.2 ± 1.5 min−1) occurred. α,β-MeATP (1 μM), a P2X agonist, caused a reduction in venular diameter (68.1 ± 9.1% of control diameter, P b 0.05) and increased SVC frequency (from 4.5 ± 1.0 min−1 to 10.3 ± 2.6 min− 1, n = 4,
Fig. 5. Effect of ACh and SIN-1 on venular spontaneous constrictions. In a suburothelial venule, ACh (1 μM) reduced basal diameter and increased the frequency of spontaneous constriction (A). A higher concentration of ACh (10 μM) further reduced venular diameter and increased the frequency of spontaneous constriction (A). In another suburothelial venule, SIN-1 (10 μM) caused a dilatation and a reduction in the frequency of spontaneous constrictions (B).
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Fig. 6. Comparative effect of ACh on arteriole and venule. In a suburothelial arteriole which had been pre-constricted with Ba2+ (500 μM), ACh (10 μM) caused a dilatation that was attenuated by LNA (10 μM) (A). In a suburothelial venule, BaCl2 (500 μM) increased the frequency of spontaneous constrictions with a reduction in the basal diameter (B). Subsequent ACh (10 μM) caused a sustained constriction (B). Dotted lines indicate basal diameter. Effects of BaCl2, ACh and LNA on the arteriolar diameter were summarized in (C). Effects of BaCl2 and ACh on the venular diameter were summarized in (D).
N = 4, P b 0.05) during 15 min application, but failed to cause either dilatation or inhibition of SVCs. 3.6. Effects of substance P and CGRP on SVCs Nerve fibers highly immunoreactive against CGRP (Fig. 8A, B) or substance P (Fig. 8E, F) were observed along arterioles, and were also detected around venules (Fig. 8A, C–F). Varicose sensory nerve fibers running along the microvasculature as well as transversely over venules (Fig. 8c–f) were observed. Substance P (100 nM) caused a transient suppression of SVCs that was followed by an increase in their frequency (from 4.5 ± 1.3 min−1 to 6.9 ± 2.6 min−1, P b 0.05) with a reduction in the basal diameter (80.0 ± 15.1% of control diameter, n = 15, N = 12, P b 0.05, Fig. 9A). In LNA-pretreated venules, substance P increased the frequency of SVCs (from 6.5 ± 3.2 min−1 to 9.5 ± 1.6 min−1, n = 3, N = 3, P b 0.05) or suppressed their generation (n = 4, N = 4) with a reduction in the basal diameter (74.6 ± 14.7% of control diameter, n = 7, N = 7, P b 0.05, Fig. 9B). CGRP (100 nM) caused a dilatation (113.2 ± 7.6% of control diameter, n = 9, N = 9, P b 0.05) and either reduced the frequency of SVCs (from 3.1 ± 0.9 min−1 to 1.2 ± 1.0 min−1, n = 3, N = 3, P b 0.05) or prevented their generation (n = 6, N = 6, Fig. 9C). In LNA-pretreated venules, CGRP was still capable of dilating the venules (122.2 ± 20.6% of control diameter, n = 8, N = 8, P b 0.05) with a reduction in SVC frequency (from 5.9 ± 3.4 min−1 to 2.7 ± 1.4 min−1, n = 3, N = 3, P b 0.05) or a prevention of their generation (n = 5, N = 5, Fig. 9D).
4. Discussion EFS evoked an acceleration of SVCs with a reduction in their basal diameter. Both excitatory effects were blocked by phentolamine, indicating that noradrenaline released from sympathetic nerves act on α-adrenoceptors to constrict suburothelial venules. In suburothelial arterioles of the rat bladder, nerve-evoked constrictions were suppressed by prazosin and RS17053, a selective α1A blocker, but not by BMY7378, an α1D blocker [15]. Thus, an elevated sympathetic nerve input to suburothelial microvasculature, that may be associated with hypertension or cardiac failure, would increase the resistance to blood circulation resulting in bladder overactivity. Therefore, it is reasonable to assume that the improvement of bladder storage function with α-adrenoceptor antagonists may be partly attributed to dilatation of the suburothelial microvasculature [22]. Such vasodilatating effects of α-adrenoceptor would be beneficial for not only improving blood supply to the tissue but also correcting tissue acidification which appears to play a critical role in the induction of urinary urgency. Since β-adrenoceptor-mediated dilatation appears to act as an endogenous anti-vasoconstriction mechanism, providing even a modest suppression of α-adrenergic action may be sufficient to improve metabolite stagnation. Consistent with nerve-evoked β-adrenoceptor-mediated dilatations of the venules, BRL37344, a β3 adrenoceptor agonist, caused venular dilatation. This inhibitory action was largely attenuated by LNA, suggesting that β-adrenoceptor stimulation may result in the release of nitric oxide from the endothelium [12]. Therefore, it is likely that β3-adrenoceptor agonists can improve bladder storage symptoms in
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Fig. 7. Effects of ATP and ADP on venular spontaneous constrictions. In a suburothelial venule, ATP (1 μM) transiently increased the frequency of spontaneous constrictions, and then prevented their generation with a dilatation of the venule (A). In the same venule that had been treated with LNA (10 μM) for 30 min, ATP caused a small constriction (B). After withdrawal of ATP, small dilatation and reduction in the frequency of spontaneous constrictions was observed (B). In another venule, ADP (1 μM) caused a transient increase in spontaneous constrictions that was followed by a cessation of spontaneous constrictions with a venular dilatation (C). In the same venule that had been treated with LNA (10 μM) for 30 min, ADP caused a small constriction that was followed by an inhibition of spontaneous constrictions upon washout (D).
overactive bladder [1] by facilitating venular drainage. One of the downstream effects of β-adrenoceptor stimulation is the release of NO from the urothelium which subsequently affects the suburothelial microvasculature [6]. On the other hand, impaired NO production in the endothelium and/or urothelium could worsen the imbalance between blood supply and venular drainage. Suburothelial arterioles expressed a weaker immunoreactivity to αSMA than did the venules. Fluo-8 fluorescence Ca2+ signals in arterioles were also less bright than those of the venules. A previous study reported the presence of accessory sheath consisting of elastic and collagenous fibers surrounding the main vesicular arteries but not veins in the rabbit bladder [19]. This provides easier access to the venular smooth muscle by non-neuronal substances released from the urothelium. Despite such low accessibility from the adventitial surface to arteriolar smooth muscle cells, EFS-evoked Ca2+ transients in arterioles had a much faster time course than those in venules, suggesting that ‘tight’ junction-like neuroeffector transmission may be predominant in the arterioles, whilst diffusion of neurotransmitters, i.e., ‘volume’ transmission, may occur in the venules. In general, ACh acts as a potent vasodilating substance by releasing NO and/or an endothelium-derived hyperpolarization factor. In suburothelial arterioles precontracted with Ba2+, which blocks inward rectifier K+ channels [17], ACh caused NO dependent vasodilatation as in most vascular beds. Strikingly, ACh caused strong constrictions of suburothelial venules, irrespective of the presence or absence of the Ba2+ pre-constrictions. Since SIN-1, a NO donor, inhibited the generation of spontaneous constrictions with a dilatation of the venules, NO itself is capable of relaxing venular smooth muscle cells, suggesting that ACh may not be able to release endothelial NO in these particular
venules. β-Adrenoceptor-stimulation, ATP or substance P caused venular dilatation by releasing NO, presumably from the endothelium but the lack of ACh-induced vasodilatation may not be due to a damaged endothelium. However, we cannot entirely exclude the possibility that the endothelium in non-perfused venules may have been impaired by hypoxia or platelet derived substances. Despite the presence of cholinergic innervation in several vascular beds, including the suburothelial microvasculature in the mouse bladder [23], a functional cholinergic innervation involving endotheliumdependent mechanisms is rather exceptional [18,29]. The nerveevoked modulation of venular contractility appears to be exclusively mediated by sympathetic nerves in suburothelial venules under the current experimental conditions. Nevertheless, ACh released from the urothelium may constrict venules resulting in stasis or stagnation of tissue metabolites, and stretch-induced and age-related increases in non-neuronal ACh release [33] have been reported. Conversely, antimuscarinic agents, the most frequently used and effective pharmacological tools for OAB treatment, may have protective effects by improving the suburothelial microcirculation. ATP, a major urothelial transmitter in afferent signaling, caused a transient acceleration of SVCs that was followed by their suppression and associated dilatation. Inhibitory effects of ATP on venular contractility were mimicked by ADP but not α,β-MeATP, suggesting that ATP acts predominantly on P2Y purinoceptors. The notion that P2X receptors mediate vascular constrictions was demonstrated when α,β-MeATP caused a vasoconstriction that was associated with an increased frequency of SVCs. In rat perfused small mesenteric arteries, luminal application of ATP caused a dilatation, whilst abluminal ATP induced a transient constriction that was followed by a sustained dilatation [31],
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Fig. 8. Immunohistochemical identification of CGRP- and substance P-containing nerves. Venules in the mucosa of the rat bladder are immunoreactive for α smooth muscle actin (αSMA) and arterioles are also weakly immunoreactive for αSMA (A). The enlarged images of a reveal that CGRP-positive nerve fibers innervate both arteriole (B) and venule (C, D). CGRP-positive nerve varicosities were indicated by arrows (B, C, D). SP-positive nerve fibers run along arteriole, and are also observed around venule (E, F). A and V indicate arteriole and venule, respectively. Scale bars: 100 μm (A, E, F), 20 μm (B, C, D).
suggesting that luminal ATP may act on endothelial P2Y receptors, and that abluminal ATP would stimulate P2X as well as P2Y receptors located on smooth muscle cells. Therefore, the endothelial target of ATP in suburothelial venules appears to be P2Y receptors resulting in NO release, although suburothelial myofibroblasts that functionally express P2Y receptor may also be involved [32]. The normal micturition reflex is mediated by Aδ afferent nerves, whilst C-fibers respond to chemical and mechanical stimulation and convey pathological urinary urgency [11]. Although precise mechanisms underlying the functional changes of bladder afferent signaling remain unknown, both anaerobic respiration subsequent to reduced oxygen supply (arteriole factor) and stagnation of metabolites (venule
factor) could result in acidification thus stimulating TRP channels on C-fibers [8,20]. Although, the physiological significance of spontaneous venular constrictions in the bladder still remains to be explored, we have recently reported spontaneous venular constrictions in the rat distal colon [24] and stomach (unpublished observations) — organs that also undergo distension. Moreover, vasomotion has been investigated in several vascular beds in vivo [7,13], and altered vasomotion has been reported in diabetes and hypertension [26]. Therefore, it is reasonable that neurohumoral modulation of SVCs may play a critical role in the physiology bladder afferent signaling. Unlike submucosal venules in the rat distal colon where repetitive EFS (at 20 Hz) evoked CGRP-mediated vasodilatation [24], EFS-induced
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Fig. 9. Effects of substance P and CGRP on venular spontaneous constrictions. In a suburothelial venule, substance P (100 nM) caused a transient suppression of spontaneous constrictions that was followed by a sustained constriction (A). In the same venule that had been treated with LNA (10 μM) for 30 min, substance P caused a sustained constriction (B). In another venule, CGRP (100 nM) caused a dilatation and prevented the generation of spontaneous constrictions (C). In the same venule that had been treated with LNA (10 μM) for 30 min, CGRP was capable of causing a dilatation with a cessation of spontaneous constrictions (D).
responses appear to be exclusively mediated by sympathetic nerves in the present experimental conditions. Nevertheless, immunohistochemical studies demonstrated that substance P- and CGRP-positive nerves ran along the suburothelial microvasculature. Thus, substance P and CGRP could be released during inflammation or tissue acidification. Substance P predominantly caused a venular constriction, whilst CGRP induced a NO-independent sustained venular relaxation, presumably by stimulating adenylate cyclase. Effects of capsaicin on venular contractility were highly variable presumably due to the release of counteracting neuropeptides; ranging from a suppression of spontaneous venular constrictions with a dilatation to a sustained vasoconstriction (unpublished observations). 4.1. Conclusions The predominant action of noradrenaline released from sympathetic nerves along suburothelial venules in the rat bladder is an α-adrenoceptor-mediated constriction, whilst a β-adrenoceptormediated relaxation appears able to counteract these vasoconstriction effects. ACh released from the urothelium may cause a stagnation of the suburothelial microcirculation, whilst the consequence of urothelial ATP release appears to be somewhat unpredictable. Substance P or CGRP released from perivascular sensory nerves may also exert an opposite modulation of the suburothelial venular drainage. Therefore, the suburothelial microcirculation may be a primary site in the etiology of overactive bladder, involving a complex interplay of a number of neurohumoral factors, and also having considerable therapeutic potential. Supplementary data to this article can be found online at http:// dx.doi.org/10.1016/j.vph.2014.01.002.
Conflict of interest The authors state no conflict of interest. Acknowledgments The authors would like to thank Dr. R. J. Lang (Monash University) for his critical reading of the manuscript. This project was supported by Grant-in-Aid for Scientific Research (B) from JSPS (No. 22390304) to H.H., and Grant-in-Aid for Challenging Exploratory Research from JSPS (No. 21659377) to H.H. References [1] Aizawa N, Homma Y, Igawa Y. Effects of mirabegron, a novel β3-adrenoceptor agonist, on primary bladder afferent activity and bladder microcontractions in rats compared with the effects of oxybutynin. Eur Urol 2012;62:1165–73. [2] Azadzoi KM, Tarcan T, Kozlowski R, Krane RJ, Siroky MB. Overactivity and structural changes in the chronically ischemic bladder. J Urol 1999;162:1768–78. [3] Azadzoi KM, Tarcan T, Siroky MB, Krane RJ. Atherosclerosis-induced chronic ischemia causes bladder fibrosis and non-compliance in the rabbit. J Urol 1999;161:1626–35. [4] Azadzoi KM, Heim VK, Tarcan T, Siroky MB. Alteration of urothelial-mediated tone in the ischemic bladder: role of eicosanoids. Neurourol Urodyn 2004;23:258–64. [5] Birder LA, Apodaca G, De Groat WC, Kanai AJ. Adrenergic- and capsaicin-evoked nitric oxide release from urothelium and afferent nerves in urinary bladder. Am J Physiol 1998;275:F226–9. [6] Birder LA, Nealen ML, Kiss S, et al. β-adrenoceptor agonists stimulate endothelial nitric oxide synthase in rat urinary bladder urothelial cells. J Neurosci 2002;22:8063–70. [7] Bouskela E, Grampp W. Spontaneous vasomotion in hamster cheek pouch arterioles in varying experimental conditions. Am J Physiol 1992;262:H478–85. [8] Brady CM, Apostolidis AN, Harper M, et al. Parallel changes in bladder suburothelial vanilloid receptor TRPV1 and pan-neuronal marker PGP9.5 immunoreactivity in patients with neurogenic detrusor overactivity after intravesical resiniferatoxin treatment. BJU Int 2004;93:770–6.
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[9] Bratslavsky G, Kogan BA, Matsumoto S, Aslan AR, Levin RM. Reperfusion injury of the rat bladder is worse than ischemia. J Urol 2003;170:2086–90. [10] Cockayne DA, Hamilton SG, Zhu QM, et al. Urinary bladder hyporeflexia and reduced pain-related behaviour in P2X3-deficient mice. Nature 2000;407:1011–5. [11] De Groat WC, Yoshimura N. Afferent nerve regulation of bladder function in health and disease. Handb Exp Pharmacol 2009;194:91–138. [12] Dessy C, Moniotte S, Ghisdal P, et al. Endothelial β3-adrenoceptors mediate vasorelaxation of human coronary microarteries through nitric oxide and endotheliumdependent hyperpolarization. Circulation 2004;110:948–54. [13] Fujii K, Heistad DD, Faraci FM. Vasomotion of basilar arteries in vivo. Am J Physiol 1999;258:H1829–34. [14] Greenland JE, Brading AF. The effect of bladder outflow obstruction on detrusor blood flow changes during the voiding cycle in conscious pigs. J Urol 2001;165:245–8. [15] Hashitani H, Takano H, Fujita K, Mitsui R, Suzuki H. Functional properties of suburothelial microvessels in the rat bladder. J Urol 2011;185:2382–91. [16] Hashitani H, Mitsui R, Shimizu Y. Functional and morphological properties of pericytes in suburothelial venules of the mouse bladder. Br J Pharmacol 2012;167:1723–36. [17] Hashitani H, Suzuki H. K+ channels which contribute to the acetylcholine-induced hyperpolarization in smooth muscle of the guinea-pig submucosal arteriole. J Physiol 1997;501:319–29. [18] Hashitani H, Windle A, Suzuki H. Neuroeffector transmission in arterioles of the guinea-pig choroid. J Physiol 1998;510:209–23. [19] Hossler FE, Monson FC. Microvasculature of the rabbit urinary bladder. Anat Rec 1995;243:438–48. [20] Holzer P. Acid-sensitive ion channels and receptors. Handb Exp Pharmacol 2009;194:283–332. [21] Jen PY, Dixon JS, Gosling JA. Immunohistochemical localization of neuromarkers and neuropeptides in human fetal and neonatal urinary bladder. Br J Urol 1995;75:230–5.
[22] Mine S, Yamamoto T, Mizuno H, et al. Effect of tamsulosin on bladder microcirculation in rat model of bladder outlet obstruction using pencil lens charge-coupled device microscopy system. Urology 2013;81:155–9. [23] Mitsui R, Hashitani H. Immunohistochemical characteristics of suburothelial microvasculature in the mouse bladder. Histochem Cell Biol 2013;140:189–200. [24] Mitsui R, Miyamoto S, Takano H, Hashitani H. Properties of submucosal venules in the rat distal colon. Br J Pharmacol 2013;170:968–77. [25] Morelli A, Filippi S, Comeglio P, et al. Acute vardenafil administration improves bladder oxygenation in spontaneously hypertensive rats. J Sex Med 2010;7:107–20. [26] Nilsson H, Aalkjaer C. Vasomotion: mechanisms and physiological importance. Mol Interv 2003;3:79–89. [27] Ouslander JG. Management of overactive bladder. N Engl J Med 2004;350:786–99. [28] Sawada N, Nomiya M, Hood B, Koslov D, Zarifpour M, Andersson KE. Protective effect of a β3-adrenoceptor agonist on bladder function in a rat model of chronic bladder ischemia. Eur Urol 2013;64:664–71. [29] Van Riper DA, Bevan JA. Electrical field stimulation-mediated relaxation of rabbit middle cerebral artery. Evidence of a cholinergic endothelium-dependent component. Circ Res 1992;70:1104–12. [30] Vlaskovska M, Kasakov L, Rong W, et al. P2X3 knock-out mice reveal a major sensory role for urothelially released ATP. J Neurosci 2001;21:5670–7. [31] Winter P, Dora KA. Spreading dilatation to luminal perfusion of ATP and UTP in rat isolated small mesenteric arteries. J Physiol 2007;582:335–47. [32] Wu C, Sui GP, Fry CH. Purinergic regulation of guinea pig suburothelial myofibroblasts. J Physiol 2004;559:231–43. [33] Yoshida M, Inadome A, Maeda Y, et al. Non-neuronal cholinergic system in human bladder urothelium. Urology 2006;67:425–30. [34] Yoshida M, Masunaga K, Nagata T, Satoji Y, Shiomi M. The effects of chronic hyperlipidemia on bladder function in myocardial infarction-prone Watanabe heritable hyperlipidemic (WHHLMI) rabbits. Neurourol Urodyn 2010;29:1350–4. [35] Yoshimura N. Lower urinary tract symptoms (LUTS) and bladder afferent activity. Neurourol Urodyn 2007;26:908–13.
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Vascular Pharmacology journal homepage: www.elsevier.com/locate/vph
Neurohumoral regulation of spontaneous constrictions in suburothelial venules of the rat urinary bladder Yuki Shimizu, Satoshi Mochizuki, Retsu Mitsui, Hikaru Hashitani ⁎ Department of Cell Physiology, Nagoya City University Graduate School of Medical Sciences, Nagoya 467-8601, Japan
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Article history: Received 18 October 2013 Received in revised form 30 December 2013 Accepted 3 January 2014 Keywords: Microcirculation Bladder Suburothelium Vasomotion Adrenergic nerve
a b s t r a c t Venules of the bladder suburothelium develop spontaneous phasic constrictions that may play a critical role in maintaining venular drainage of tissue metabolites. We aimed to investigate neurohumoral regulation of the spontaneous venular constrictions (SVCs). Changes in venular diameter of the rat bladder suburothelium were monitored using a video tracking system, whilst the effects of electrical field stimulation (EFS) and bathapplied bioactive substances were investigated. The innervation of the suburothelial microvasculature was examined by immunohistochemistry. EFS (10 Hz for 30 s) induced an increase in the frequency of SVCs that was prevented by phentolamine (1 μM). In phentolamine-pretreated venules, EFS suppressed SVCs with a venular dilatation in a manner attenuated by propranolol (1 μM) or L-nitro arginine (LNA, 10 μM). BRL37344 (1 μM), a β3 adrenoceptor agonist, dilated venules and reduced the frequency of SVCs in an LNA-sensitive manner. ACh (1–10 μM) increased the frequency of SVCs. ATP (1 μM) transiently constricted venules and then caused LNA-sensitive cessation of SVCs associated with a dilatation. Substance P (100 nM) caused a venular constriction, whilst calcitonin gene related peptide (CGRP, 100 nM) caused a dilatation of venules and suppression of SVCs that were not inhibited by LNA. Immunohistochemical staining demonstrated sympathetic as well as substance P- and CGRP-containing nerves running along the venules. Spontaneous constrictions of suburothelial venules are accelerated by sympathetic α-adrenergic stimulation, but suppressed upon β-adrenergic stimulation. In addition, suburothelial venular constrictions appear to be modulated by several bioactive substances that could be released from urothelium or suburothelial sensory nerves. © 2014 Elsevier Inc. All rights reserved.
1. Introduction Overactive bladder (OAB) is a highly prevalent disorder that increases with age in both sexes. Its cause or etiology appears to be complex and multifactorial [27]. Considering the current diagnostic criteria of over active bladder, i.e., urinary urgency, enhanced signal transmission from urothelium to afferent nerves may be a primary cause of overactive bladder symptoms [35]. Normal bladder filling sensation is mediated by stretch-dependent release of ATP from urothelium that acts upon P2X3 purinoceptors located on sensory nerve endings [10,30]. ATP also acts on suburothelial interstitial cells [32] or detrusor smooth muscle cells that may modulate urothelium-afferent nerve signal transmission. Thus excessive releases of ATP could result in urinary urgency. Additionally, other bioactive substances released from the urothelium, e.g., acetylcholine [33] and nitric oxide [5], or from sensory nerves [21], e.g., substance P and CGRP, may also modulate afferent nerve activity.
Abbreviations: LNA, N-nitro-L-arginine; SIN-1, 3-(4-Morpholinyl)sydnonimine; CGRP, calcitonin gene-related peptide; EFS, electrical filed stimulation; SVC, spontaneous venular constriction; OAB, overactive bladder. ⁎ Corresponding author. Tel.: +81 52 8538131; fax: +81 52 8421538. E-mail address: [email protected] (H. Hashitani). 1537-1891/$ – see front matter © 2014 Elsevier Inc. All rights reserved. http://dx.doi.org/10.1016/j.vph.2014.01.002
The suburothelial microcirculation is critical in maintaining bladder cell function. In the normal bladder, blood flow is well maintained during the filling phase, and only transiently decreases during voiding [14]. This maintenance primarily relies on the high compliance properties of the bladder wall that allow low pressure storage of urine. However the intrinsic properties of the microvasculature including their winding arrangement and spontaneous venular constrictions (SVCs) [15,16] may also be beneficial in maintaining bladder microcirculation during storage phase. It has been proposed that overactive bladder results from ischemia and/or reperfusion [9] associated with bladder outlet obstruction, aging or atherosclerosis [2,3,34]. Indeed, alterations in urothelial mediated regulation in ischemic bladders result in detrusor instability [2–4]. Previous studies focused on the arteries/arterioles in terms of their function in supplying oxygen and nutrients to the bladder, whilst the role of venules in regulating the bladder microcirculation has garnered less attention. Of studies that examine bladder venules, it is suggested that diminished venular drainage can result in tissue metabolite accumulation and contribute to ‘urinary urgency’ upon acidification-induced stimulation of TRP channels at sensory nerve endings [8,20]. Thus it is reasonable to assume that improvement of bladder storage symptoms with α-adrenoceptor antagonists [22] or PDE5 inhibitors [25] may be attributed to their vasodilator action on the suburothelial microvasculature.
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β3-Adrenoceptor agonists may also exert an influence by improving the microcirculation within the bladder wall [1]. For example, mirabegron, a β3-adrenoceptor agonist, reduces bladder hyperactivity in a rat model of chronic bladder ischemia [28]. The suburothelial microvasculature runs within the connective tissue layer just beneath the urothelium, where both efferent and afferent nerves are abundant, suggesting their function could readily be affected by neurohumoral substances released from nerves as well as the urothelium. We aimed to investigate the neurohumoral regulation of SVCs in the rat bladder since any imbalance between excitatory and inhibitory vasoactive substances may result in suburothelial microcirculation dysfunction. Agents that improve the microcirculation are potential tools for the treatment or even prophylactic treatment of overactive bladder. 2. Materials and methods
Specimens were incubated with PBS containing 0.3% Triton X-100 for 10 min, incubated with Block Ace (Dainippon Seiyaku) for 20 min and incubated with primary antibodies diluted in PBS containing 2% bovine serum albumin and 0.3% Triton X-100 for 4 days at 4 °C. Primary antibodies used in the present study were as follows: mouse monoclonal antibody for α smooth muscle actin (αSMA; 1:500, clone 1A4, Sigma), rabbit anti tyrosine hydroxylase (TH) antibody (1:1000, Millipore), rabbit anti-SP antibody (1:1000, Immunostar) and rabbit anti-CGRP antibody (1:50, Progen Biotechnik). Specimens were then incubated with biotinylated swine anti-rabbit IgG antibody (1:300, Dako) for 30 min at room temperature. Specimens were incubated with Alexa 488-conjugated streptavidin (10 μg/ml, Jackson ImmunoResearch) and Cy-3-conjugated goat anti-mouse IgG antibody (2.5 μg/ml, Millipore) for 2 h at room temperature and coverslipped with a fluorescence mounting medium (Dako). Whole mounts were examined using a confocal laser scanning microscope (LSM 5 PASCAL, Zeiss).
2.1. Animal and ethical approval
2.6. Solutions and drugs
Male Wistar rats 6 to 8 weeks old were used throughout this study. They were housed, cared for and acclimatized (before the experiments). For experiments, rats were anesthetized and exsanguinated by decapitation according to procedures approved by the Nagoya City University Medical School Experimental Animal Committee.
The composition of PSS was (in mM): NaCl, 120; KCl, 4.7; MgCl2, 1.2; CaCl2, 2.5; NaHCO3, 15.5; KH2PO4, 1.2 and glucose, 11.5. The pH of PSS was 7.2 when bubbled with 95% O2 and 5% CO2, and the measured pH of the recording bath was approximately 7.4. The drugs used were acetylcholine (ACh), ADP, ATP, αβ-methylene ATP (αβ-MeATP), BaCl2, BRL37344, human CGRP, phentolamine, propranolol, N-ω-nitro-L-arginine (LNA), 3-(4-morpholinyl)sydnonimine (SIN-1), substance P and tetrodotoxin. All drugs were dissolved in distilled water except SIN-1 which was dissolved in ethanol. The final concentration of solvents in PSS did not exceed 1:1000.
2.2. Tissue preparation The bladder was removed, cut open and pinned in a dissecting dish with the urothelial side uppermost. The mucosal layer was dissected away from the detrusor smooth muscle layer, and the urothelium was carefully removed using ophthalmology scissors leaving the suburothelial layer. 2.3. Video imaging Suburothelial preparations were superfused with warmed (36 °C) physiological salt solution (PSS) at a constant flow rate (about 2 ml/ min). Venules were readily distinguished from arterioles by their larger diameters and thinner walls as well as their regularly occurring SVCs. Changes in the diameter of venules were recorded with a video camera, and analyzed using Diamtrak, video edge detection software. Electrical field stimulation (EFS) was applied to preparations by passing brief currents (10 Hz, pulse width 50 μs) between a pair of platinum electrodes in the recording chamber. The neural selectivity of evoked responses was confirmed by their sensitivity to tetrodotoxin (1 μM). 2.4. Intracellular calcium imaging Suburothelial preparations were incubated in low Ca 2 + PSS ([Ca2 +] o = 0.1 mM) containing 1 μM fluo-8 AM (AAT Bioquest, Sunnyvale, CA, USA) and cremphor EL (0.01%, Sigma) for 30 min at 35 °C. Following incubation, preparations were superfused with dye-free PSS and illuminated at 495 nm with fluorescence emissions (above 515 nm) captured as described previously [10,11]. Relative amplitudes of Ca2+ transients were expressed as ΔFt/F0 = (Ft − F0) / F0, where Ft is the fluorescence generated by an event, and baseline F0 is the basal fluorescence. 2.5. Immunohistochemistry Suburothelial whole mount preparations were immersed in fixative containing 2% formaldehyde and 15% saturated picric acid in 0.1 M phosphate buffer (pH 7.4) for 10 min, and then further immersed in the same fixative overnight at 4 °C. The fixed specimens were washed in dimethyl sulfoxide (DMSO) and then in PBS.
2.7. Calculations and statistics Measured values are shown as the mean ± SD. Statistical significance was tested using the paired t test with P b 0.05 considered significant. The number of tissues is denoted ‘n’, whilst the number of animals is denoted ‘N’. Peak amplitude of SVCs was measured as the value from the basal diameter to the peak of constrictions. Frequency was calculated as an average during 3 min either prior to (control) or during drug application, or was calculated during EFS. 3. Results 3.1. General observations Consistent with our previous report [15], suburothelial venules developed spontaneous phasic constrictions. In 54 suburothelial venules (n = 54, N = 48), SVCs were associated with a transient reduction in their diameter to 78.5 ± 13.2% of their resting diameter (70.9 ± 24.9 μm) and occurred at a frequency of 4.7 ± 1.7 min− 1. During preliminary experiments, a substantial delay between EFS and the onset of venular constrictions were evident. Therefore, the time course of nerve-evoked responses in venules and arterioles was compared by visualizing their Ca2+ transients. EFS (10 Hz for 1 s) triggered a prompt Ca2+ transient in nerve fibers that was immediately followed by Ca2+ transients in the arteriolar smooth muscle cells with a delay of 0.57 ± 0.04 s (n = 5, N = 4, Fig. 1A, B). Subsequently, EFSinduced Ca2+ transients in venular smooth muscle cells were observed with a delay of 2.1 ± 0.17 s (n = 5, N = 4, Fig. 1A, B, Supplement movie 1). Suburothelial venules were immunoreactive for α smooth muscle actin (αSMA, Fig. 1C a, b, d), whilst arterioles showed relatively weak αSMA immunoreactivity (Fig. 1Ca, b). Both arterioles (Fig. 1Cb, c) and venules (Fig. 1Cb, c) were innervated, and varicose sympathetic nerve fibers immunopositive for tyrosine hydroxylase (TH) run along the microvasculature as well as transversely over venules (Fig. 1Cd, e).
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3.2. Effects of EFS on SVCs In suburothelial venules, EFS (10 Hz for 30 s) induced an increase in the frequency of SVCs (from 4.7 ± 1.4 min− 1 to 11.3 ± 2.4 min− 1,
P b 0.05) associated with a sustained reduction in their diameter (76.9 ± 13.7% of control diameter, n = 15, N = 15, P b 0.05, Fig. 2A). These excitatory effects were prevented by phentolamine (1 μM, n = 12, N = 12), such that subsequent EFS induced a venular dilatation
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Fig. 2. Neural regulation of spontaneous venular constrictions. In a suburothelial venule, EFS (under bar, 10 Hz for 30 s) increased the frequency of spontaneous constrictions and caused a sustained constriction (A). In the same venule that had been treated with phentolamine (10 μM) for 20 min, EFS reduced the frequency of spontaneous constrictions with a small dilatation (B). Subsequent propranolol prevented the inhibitory effects of EFS (C). Dotted lines indicate basal diameter. Effects of EFS and phentolamine on the venular diameter and the frequency of spontaneous constrictions were summarized in (C) and (D), respectively.
(112.2 ± 4.2% of control diameter, n = 5, N = 5, P b 0.05, Fig. 2B, D) with a reduction in the frequency of SVCs (from 5.4 ± 1.4 min−1 to 2.6 ± 0.4 min−1, P b 0.05, Fig. 2B, E) that was prevented by propranolol (1 μM, n = 5, N = 5, Fig. 2C). In phentolamine-pretreated venules, EFS-induced venular dilatation was accompanied with either a cessation of SVCs (n = 4, N = 4, Fig. 3A) or a reduction in their frequency (from 5.2 ± 1.1 min− 1 to 2.4 ± 0.7 min−1, n = 10, N = 10, P b 0.05). LNA (10 μM) increased the frequency of SVCs (from 5.4 ± 0.9 min− 1 to 8.1 ± 1.5 min−1, n = 7, N = 7, P b 0.05), and caused a sustained reduction in the basal
diameter (81.9 ± 7.5% of control diameter, n = 7, N = 7, P b 0.05) associated with a suppression of SVC amplitude. In preparations which had been treated with LNA for 30 min, EFS failed to induce venular dilatation (Fig. 3B, C, D). BRL37344 (1 μM), a β3-adrenoceptor agonist, reduced the frequency of SVCs (from 4.7 ± 0.6 min − 1 to 2.5 ± 0.6 min − 1 , n = 6, N = 6, P b 0.05, Fig. 4A) with a venular dilatation (125.7 ± 13.6% of control diameter, P b 0.05). In LNA-pretreated preparations, BRL37344 failed to suppress SVCs (n = 5, N = 5, Fig. 4B, C, D).
Fig. 1. Sympathetic innervation to venules of the bladder suburothelium. (A) Sequential fluo-8 fluorescence Ca2+ images with a frame interval of 770 ms showing EFS (10 Hz for 1 s)induced Ca2+ transient in a nerve fiber (ROI 1), arteriolar smooth muscle (ROI 2) and venular smooth muscle (ROI 3) in a bladder suburothelial preparation. (B) EFS (under bar, 10 Hz for 1 s) evoked a Ca2+ transient in a nerve fiber (black) that was immediately followed by a Ca2+ transient in an arteriole (magenta), and subsequently induced a Ca2+ transient in a venule (blue). (C) Immunoreactivity for α-smooth muscle actin (αSMA) revealed a suburothelial arteriole (A) and venule (V) in the rat bladder (a). Varicose sympathetic nerve fibers immunopositive for tyrosine hydroxylase (TH) innervated both the arteriole and venule (b, c). Immunoreactivity for αSMA in the venule is brighter than that of the arteriole. Several varicose sympathetic fibers projecting to suburothelial venule (V) were detected in another specimen (d, e). Scale bars indicate 50 μm (a) and 20 μm (d), respectively.
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Fig. 3. Effect of LNA on β-adrenergic relaxation. In a suburothelial venule that had been treated with phentolamine (10 μM), EFS (under bar, 10 Hz for 30 s) evoked a venular dilatation with a cessation of spontaneous constrictions (A). In the same venule that had been treated with LNA (10 μM) for 30 min. EFS-induced inhibition of spontaneous constrictions was largely attenuated (B). Dotted lines indicate basal diameter. Effects of EFS and LNA on the venular diameter and the frequency of spontaneous constrictions were summarized in (C) and (D), respectively.
3.3. Effect of ACh and SIN-1 on SVCs Since urothelium-derived ACh may affect suburothelial microvasculature function, the effects of bath-applied ACh on venular contractility were examined. Unexpectedly, ACh (1 μM) increased the frequency of SVCs (from 4.5 ± 1.8 min−1 to 5.8 ± 2.1 min−1, P b 0.05) and caused a reduction in the basal diameter of the venules (89.2 ± 12.4% of control diameter, n = 14, N = 10, P b 0.05, Fig. 5A). A higher concentration of ACh (10 μM) further increased the frequency of SVCs to 6.6 ± 1.9 min− 1, and reduced venular diameter (83.7 ± 15.1% of control diameter, Fig. 5A). Vasorelaxant action of nitric oxide (NO), a predominant endotheliumderived relaxing factor, in suburothelial venules, was evaluated using SIN-1, a NO donor. SIN-1 (1 μM) caused a marked venular dilatation (117 ± 12.7% of control diameter, n = 9, N = 9, P b 0.05), and either reduced the frequency of SVCs (from 3.7 ± 0.5 min− 1 to 2.4 ± 0.75 min−1, n = 3, N = 3, P b 0.05, Fig. 5B) or prevented their generation (n = 6, N = 6). 3.4. Comparative effects of ACh on arteriolar and venular contractility One might argue that the lack of ACh-induced dilatation of the venules may be attributed to damages to their endothelium. Thus effects of ACh on suburothelial arterioles running along the venules were investigated. In suburothelial arterioles, Ba2+ (500 μM), a blocker for inward rectifier potassium channels [23], caused a sustained reduction in the basal diameter (61.9 ± 7.2% of control diameter, n = 10, N = 9, P b 0.05). Subsequent ACh (10 μM) caused a dilatation of the Ba2 +-contracted
arterioles to 97.8 ± 11.7% of control diameter. ACh-induced dilatations were partially reversed by LNA (10 μM, 68.2 ± 17.2% of control diameter, n = 6, N = 6, Fig. 6A, C), suggesting that ACh was capable of releasing NO from the arteriolar endothelium. In suburothelial venules, Ba2+ (500 μM), increased the frequency of SVCs (from 3.5 ± 1.1 min− 1 to 5.4 ± 2.3 min− 1, n = 7, N = 7, P b 0.05) and often induced long lasting constrictions which appeared to be composed of fused SVCs with a reduction in the basal diameter (94.0 ± 4.2% of control diameter, P b 0.05, Fig. 6B). ACh (10 μM) caused a sustained constriction (82.8 ± 14.8% of control diameter, n = 10, N = 10, P b 0.05, Fig. 6B, D). During the sustained constrictions, individual SVCs were fused and undistinguishable (n = 8, N = 8) or their frequency was further increased (6.3 ± 0.6 min−1, n = 2, N = 2). 3.5. Effects of ATP on SVCs ATP (1 μM), a major urothelial transmitter, transiently increased the frequency of SVCs (from 4.9 ± 0.7 min− 1 to 11.5 ± 2.3 min−1, P b 0.05) with a reduction in the basal diameter (78.5 ± 9.2% of control diameter, P b 0.05), and then prevented SVCs with a dilatation of venular diameter (123.6 ± 15.1% of control diameter, n = 8, N = 8, P b 0.05, Fig. 7A). In LNA-pre-treated venules, ATP caused a small constriction (88.9 ± 5.4% of control diameter, n = 5, N = 5, P b 0.05, Fig. 7B). Upon withdrawal of ATP, there was a small dilatation (105 ± 2.2% of control diameter, P b 0.05) and reduction in the frequency of SVCs (from 10.4 ± 2.5 min−1 to 8.9 ± 2.1 min−1, P b 0.05). ADP (1 μM), a P2Y agonist, caused a transient increase in SVCs (from 4.9 ± 0.9 min− 1 to 10.1 ± 1.1 min− 1, P b 0.05) with a reduction in venular diameter (80.2 ± 6.6% of control diameter, P b 0.05),
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Fig. 4. Effect of BRL 37344 on venular spontaneous constrictions. In a suburothelial venule, BRL 37344 (1 μM) caused a dilatation associated with a reduction in the frequency of spontaneous constrictions (A). In the same venule that had been treated with LNA (10 μM) for 30 min. BRL 37344 (1 μM) failed to suppress spontaneous constrictions (B). Dotted lines indicate basal diameter. Effects of BRL 37344 and LNA on the venular diameter and the frequency of spontaneous constrictions were summarized in (C) and (D), respectively.
and then caused a cessation of SVCs with a dilatation of venules (121.8 ± 7.5% of control diameter, n = 5, N = 5, P b 0.05, Fig. 7C). In LNA-pretreated venules, ADP caused a small constriction (92.6 ± 6.9% of control diameter, n = 3, N = 3, P b 0.05, Fig. 7D). After withdrawal of ADP, a small dilatation (105.2 ± 1.1% of control diameter, P b 0.05)
and reduction in the frequency of SVCs (from 9.7 ± 0.6 min− 1 to 7.2 ± 1.5 min−1) occurred. α,β-MeATP (1 μM), a P2X agonist, caused a reduction in venular diameter (68.1 ± 9.1% of control diameter, P b 0.05) and increased SVC frequency (from 4.5 ± 1.0 min−1 to 10.3 ± 2.6 min− 1, n = 4,
Fig. 5. Effect of ACh and SIN-1 on venular spontaneous constrictions. In a suburothelial venule, ACh (1 μM) reduced basal diameter and increased the frequency of spontaneous constriction (A). A higher concentration of ACh (10 μM) further reduced venular diameter and increased the frequency of spontaneous constriction (A). In another suburothelial venule, SIN-1 (10 μM) caused a dilatation and a reduction in the frequency of spontaneous constrictions (B).
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Fig. 6. Comparative effect of ACh on arteriole and venule. In a suburothelial arteriole which had been pre-constricted with Ba2+ (500 μM), ACh (10 μM) caused a dilatation that was attenuated by LNA (10 μM) (A). In a suburothelial venule, BaCl2 (500 μM) increased the frequency of spontaneous constrictions with a reduction in the basal diameter (B). Subsequent ACh (10 μM) caused a sustained constriction (B). Dotted lines indicate basal diameter. Effects of BaCl2, ACh and LNA on the arteriolar diameter were summarized in (C). Effects of BaCl2 and ACh on the venular diameter were summarized in (D).
N = 4, P b 0.05) during 15 min application, but failed to cause either dilatation or inhibition of SVCs. 3.6. Effects of substance P and CGRP on SVCs Nerve fibers highly immunoreactive against CGRP (Fig. 8A, B) or substance P (Fig. 8E, F) were observed along arterioles, and were also detected around venules (Fig. 8A, C–F). Varicose sensory nerve fibers running along the microvasculature as well as transversely over venules (Fig. 8c–f) were observed. Substance P (100 nM) caused a transient suppression of SVCs that was followed by an increase in their frequency (from 4.5 ± 1.3 min−1 to 6.9 ± 2.6 min−1, P b 0.05) with a reduction in the basal diameter (80.0 ± 15.1% of control diameter, n = 15, N = 12, P b 0.05, Fig. 9A). In LNA-pretreated venules, substance P increased the frequency of SVCs (from 6.5 ± 3.2 min−1 to 9.5 ± 1.6 min−1, n = 3, N = 3, P b 0.05) or suppressed their generation (n = 4, N = 4) with a reduction in the basal diameter (74.6 ± 14.7% of control diameter, n = 7, N = 7, P b 0.05, Fig. 9B). CGRP (100 nM) caused a dilatation (113.2 ± 7.6% of control diameter, n = 9, N = 9, P b 0.05) and either reduced the frequency of SVCs (from 3.1 ± 0.9 min−1 to 1.2 ± 1.0 min−1, n = 3, N = 3, P b 0.05) or prevented their generation (n = 6, N = 6, Fig. 9C). In LNA-pretreated venules, CGRP was still capable of dilating the venules (122.2 ± 20.6% of control diameter, n = 8, N = 8, P b 0.05) with a reduction in SVC frequency (from 5.9 ± 3.4 min−1 to 2.7 ± 1.4 min−1, n = 3, N = 3, P b 0.05) or a prevention of their generation (n = 5, N = 5, Fig. 9D).
4. Discussion EFS evoked an acceleration of SVCs with a reduction in their basal diameter. Both excitatory effects were blocked by phentolamine, indicating that noradrenaline released from sympathetic nerves act on α-adrenoceptors to constrict suburothelial venules. In suburothelial arterioles of the rat bladder, nerve-evoked constrictions were suppressed by prazosin and RS17053, a selective α1A blocker, but not by BMY7378, an α1D blocker [15]. Thus, an elevated sympathetic nerve input to suburothelial microvasculature, that may be associated with hypertension or cardiac failure, would increase the resistance to blood circulation resulting in bladder overactivity. Therefore, it is reasonable to assume that the improvement of bladder storage function with α-adrenoceptor antagonists may be partly attributed to dilatation of the suburothelial microvasculature [22]. Such vasodilatating effects of α-adrenoceptor would be beneficial for not only improving blood supply to the tissue but also correcting tissue acidification which appears to play a critical role in the induction of urinary urgency. Since β-adrenoceptor-mediated dilatation appears to act as an endogenous anti-vasoconstriction mechanism, providing even a modest suppression of α-adrenergic action may be sufficient to improve metabolite stagnation. Consistent with nerve-evoked β-adrenoceptor-mediated dilatations of the venules, BRL37344, a β3 adrenoceptor agonist, caused venular dilatation. This inhibitory action was largely attenuated by LNA, suggesting that β-adrenoceptor stimulation may result in the release of nitric oxide from the endothelium [12]. Therefore, it is likely that β3-adrenoceptor agonists can improve bladder storage symptoms in
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Fig. 7. Effects of ATP and ADP on venular spontaneous constrictions. In a suburothelial venule, ATP (1 μM) transiently increased the frequency of spontaneous constrictions, and then prevented their generation with a dilatation of the venule (A). In the same venule that had been treated with LNA (10 μM) for 30 min, ATP caused a small constriction (B). After withdrawal of ATP, small dilatation and reduction in the frequency of spontaneous constrictions was observed (B). In another venule, ADP (1 μM) caused a transient increase in spontaneous constrictions that was followed by a cessation of spontaneous constrictions with a venular dilatation (C). In the same venule that had been treated with LNA (10 μM) for 30 min, ADP caused a small constriction that was followed by an inhibition of spontaneous constrictions upon washout (D).
overactive bladder [1] by facilitating venular drainage. One of the downstream effects of β-adrenoceptor stimulation is the release of NO from the urothelium which subsequently affects the suburothelial microvasculature [6]. On the other hand, impaired NO production in the endothelium and/or urothelium could worsen the imbalance between blood supply and venular drainage. Suburothelial arterioles expressed a weaker immunoreactivity to αSMA than did the venules. Fluo-8 fluorescence Ca2+ signals in arterioles were also less bright than those of the venules. A previous study reported the presence of accessory sheath consisting of elastic and collagenous fibers surrounding the main vesicular arteries but not veins in the rabbit bladder [19]. This provides easier access to the venular smooth muscle by non-neuronal substances released from the urothelium. Despite such low accessibility from the adventitial surface to arteriolar smooth muscle cells, EFS-evoked Ca2+ transients in arterioles had a much faster time course than those in venules, suggesting that ‘tight’ junction-like neuroeffector transmission may be predominant in the arterioles, whilst diffusion of neurotransmitters, i.e., ‘volume’ transmission, may occur in the venules. In general, ACh acts as a potent vasodilating substance by releasing NO and/or an endothelium-derived hyperpolarization factor. In suburothelial arterioles precontracted with Ba2+, which blocks inward rectifier K+ channels [17], ACh caused NO dependent vasodilatation as in most vascular beds. Strikingly, ACh caused strong constrictions of suburothelial venules, irrespective of the presence or absence of the Ba2+ pre-constrictions. Since SIN-1, a NO donor, inhibited the generation of spontaneous constrictions with a dilatation of the venules, NO itself is capable of relaxing venular smooth muscle cells, suggesting that ACh may not be able to release endothelial NO in these particular
venules. β-Adrenoceptor-stimulation, ATP or substance P caused venular dilatation by releasing NO, presumably from the endothelium but the lack of ACh-induced vasodilatation may not be due to a damaged endothelium. However, we cannot entirely exclude the possibility that the endothelium in non-perfused venules may have been impaired by hypoxia or platelet derived substances. Despite the presence of cholinergic innervation in several vascular beds, including the suburothelial microvasculature in the mouse bladder [23], a functional cholinergic innervation involving endotheliumdependent mechanisms is rather exceptional [18,29]. The nerveevoked modulation of venular contractility appears to be exclusively mediated by sympathetic nerves in suburothelial venules under the current experimental conditions. Nevertheless, ACh released from the urothelium may constrict venules resulting in stasis or stagnation of tissue metabolites, and stretch-induced and age-related increases in non-neuronal ACh release [33] have been reported. Conversely, antimuscarinic agents, the most frequently used and effective pharmacological tools for OAB treatment, may have protective effects by improving the suburothelial microcirculation. ATP, a major urothelial transmitter in afferent signaling, caused a transient acceleration of SVCs that was followed by their suppression and associated dilatation. Inhibitory effects of ATP on venular contractility were mimicked by ADP but not α,β-MeATP, suggesting that ATP acts predominantly on P2Y purinoceptors. The notion that P2X receptors mediate vascular constrictions was demonstrated when α,β-MeATP caused a vasoconstriction that was associated with an increased frequency of SVCs. In rat perfused small mesenteric arteries, luminal application of ATP caused a dilatation, whilst abluminal ATP induced a transient constriction that was followed by a sustained dilatation [31],
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Fig. 8. Immunohistochemical identification of CGRP- and substance P-containing nerves. Venules in the mucosa of the rat bladder are immunoreactive for α smooth muscle actin (αSMA) and arterioles are also weakly immunoreactive for αSMA (A). The enlarged images of a reveal that CGRP-positive nerve fibers innervate both arteriole (B) and venule (C, D). CGRP-positive nerve varicosities were indicated by arrows (B, C, D). SP-positive nerve fibers run along arteriole, and are also observed around venule (E, F). A and V indicate arteriole and venule, respectively. Scale bars: 100 μm (A, E, F), 20 μm (B, C, D).
suggesting that luminal ATP may act on endothelial P2Y receptors, and that abluminal ATP would stimulate P2X as well as P2Y receptors located on smooth muscle cells. Therefore, the endothelial target of ATP in suburothelial venules appears to be P2Y receptors resulting in NO release, although suburothelial myofibroblasts that functionally express P2Y receptor may also be involved [32]. The normal micturition reflex is mediated by Aδ afferent nerves, whilst C-fibers respond to chemical and mechanical stimulation and convey pathological urinary urgency [11]. Although precise mechanisms underlying the functional changes of bladder afferent signaling remain unknown, both anaerobic respiration subsequent to reduced oxygen supply (arteriole factor) and stagnation of metabolites (venule
factor) could result in acidification thus stimulating TRP channels on C-fibers [8,20]. Although, the physiological significance of spontaneous venular constrictions in the bladder still remains to be explored, we have recently reported spontaneous venular constrictions in the rat distal colon [24] and stomach (unpublished observations) — organs that also undergo distension. Moreover, vasomotion has been investigated in several vascular beds in vivo [7,13], and altered vasomotion has been reported in diabetes and hypertension [26]. Therefore, it is reasonable that neurohumoral modulation of SVCs may play a critical role in the physiology bladder afferent signaling. Unlike submucosal venules in the rat distal colon where repetitive EFS (at 20 Hz) evoked CGRP-mediated vasodilatation [24], EFS-induced
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Fig. 9. Effects of substance P and CGRP on venular spontaneous constrictions. In a suburothelial venule, substance P (100 nM) caused a transient suppression of spontaneous constrictions that was followed by a sustained constriction (A). In the same venule that had been treated with LNA (10 μM) for 30 min, substance P caused a sustained constriction (B). In another venule, CGRP (100 nM) caused a dilatation and prevented the generation of spontaneous constrictions (C). In the same venule that had been treated with LNA (10 μM) for 30 min, CGRP was capable of causing a dilatation with a cessation of spontaneous constrictions (D).
responses appear to be exclusively mediated by sympathetic nerves in the present experimental conditions. Nevertheless, immunohistochemical studies demonstrated that substance P- and CGRP-positive nerves ran along the suburothelial microvasculature. Thus, substance P and CGRP could be released during inflammation or tissue acidification. Substance P predominantly caused a venular constriction, whilst CGRP induced a NO-independent sustained venular relaxation, presumably by stimulating adenylate cyclase. Effects of capsaicin on venular contractility were highly variable presumably due to the release of counteracting neuropeptides; ranging from a suppression of spontaneous venular constrictions with a dilatation to a sustained vasoconstriction (unpublished observations). 4.1. Conclusions The predominant action of noradrenaline released from sympathetic nerves along suburothelial venules in the rat bladder is an α-adrenoceptor-mediated constriction, whilst a β-adrenoceptormediated relaxation appears able to counteract these vasoconstriction effects. ACh released from the urothelium may cause a stagnation of the suburothelial microcirculation, whilst the consequence of urothelial ATP release appears to be somewhat unpredictable. Substance P or CGRP released from perivascular sensory nerves may also exert an opposite modulation of the suburothelial venular drainage. Therefore, the suburothelial microcirculation may be a primary site in the etiology of overactive bladder, involving a complex interplay of a number of neurohumoral factors, and also having considerable therapeutic potential. Supplementary data to this article can be found online at http:// dx.doi.org/10.1016/j.vph.2014.01.002.
Conflict of interest The authors state no conflict of interest. Acknowledgments The authors would like to thank Dr. R. J. Lang (Monash University) for his critical reading of the manuscript. This project was supported by Grant-in-Aid for Scientific Research (B) from JSPS (No. 22390304) to H.H., and Grant-in-Aid for Challenging Exploratory Research from JSPS (No. 21659377) to H.H. References [1] Aizawa N, Homma Y, Igawa Y. Effects of mirabegron, a novel β3-adrenoceptor agonist, on primary bladder afferent activity and bladder microcontractions in rats compared with the effects of oxybutynin. Eur Urol 2012;62:1165–73. [2] Azadzoi KM, Tarcan T, Kozlowski R, Krane RJ, Siroky MB. Overactivity and structural changes in the chronically ischemic bladder. J Urol 1999;162:1768–78. [3] Azadzoi KM, Tarcan T, Siroky MB, Krane RJ. Atherosclerosis-induced chronic ischemia causes bladder fibrosis and non-compliance in the rabbit. J Urol 1999;161:1626–35. [4] Azadzoi KM, Heim VK, Tarcan T, Siroky MB. Alteration of urothelial-mediated tone in the ischemic bladder: role of eicosanoids. Neurourol Urodyn 2004;23:258–64. [5] Birder LA, Apodaca G, De Groat WC, Kanai AJ. Adrenergic- and capsaicin-evoked nitric oxide release from urothelium and afferent nerves in urinary bladder. Am J Physiol 1998;275:F226–9. [6] Birder LA, Nealen ML, Kiss S, et al. β-adrenoceptor agonists stimulate endothelial nitric oxide synthase in rat urinary bladder urothelial cells. J Neurosci 2002;22:8063–70. [7] Bouskela E, Grampp W. Spontaneous vasomotion in hamster cheek pouch arterioles in varying experimental conditions. Am J Physiol 1992;262:H478–85. [8] Brady CM, Apostolidis AN, Harper M, et al. Parallel changes in bladder suburothelial vanilloid receptor TRPV1 and pan-neuronal marker PGP9.5 immunoreactivity in patients with neurogenic detrusor overactivity after intravesical resiniferatoxin treatment. BJU Int 2004;93:770–6.
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