Basic and Translational Science Effects of Ischemia and Oxidative Stress on Bladder Purinoceptors Expression Qi Zhang, Mike Siroky, Jing-Hua Yang, Zuohui Zhao, and Kazem Azadzoi OBJECTIVE
MATERIALS AND METHODS
RESULTS
CONCLUSION
To study the effects of chronic ischemia on bladder purinoceptors. A close correlation between bladder ischemia and lower urinary tract symptoms has been reported. Purinoceptors contribute to important aspects of bladder function including sensation, neural signaling, and voiding contraction. Our goal was to examine purinoceptors expression in the ischemic overactive bladder. Moderate bladder ischemia was produced in rabbits by creating bilateral iliac artery atherosclerosis. After 8 weeks, bladder blood flow was measured, and cystometrograms were obtained. Bladder tissues from 8-week ischemic and age-matched control bladders were processed for the analysis of oxidative stress markers, P2X and P2Y purinoceptors expression, and transmission electron microscopy. Arterial atherosclerosis significantly decreased bladder blood flow. Markers of oxidative stress characterized by increased levels of advanced oxidation protein products and malondialdehyde were evident in the ischemic bladder tissues. Chronic ischemia and oxidative stress decreased the bladder capacity and increased spontaneous bladder contractions. Bladder pressure at micturition and intravesical pressure rise during contractions tended to be greater in the ischemic bladder but did not reach significance. Transmission electron microscopy showed smooth muscle cell and microvasculature structural damage and diffuse fibrosis. These changes in the ischemic bladder were associated with significant increases in purinoceptors P2X1, P2X2, P2X3, P2X4, P2X5, and P2X7 expression. The P2Y isoforms were not expressed in the rabbit bladder. Structural and functional changes in the chronically ischemic bladder were associated with upregulation of P2X receptor isoforms. Increased P2X expression may play a role in ischemia-induced bladder overactivity and noncompliance. UROLOGY 84: 1249.e1e1249.e7, 2014. Published by Elsevier Inc.
P
elvic arterial insufficiency may contribute to agingassociated bladder dysfunction and lower urinary tract symptoms (LUTS).1-5 In experimental models, moderate ischemia resulted in bladder overactivity, whereas severe ischemia diminished smooth muscle contractile responses.1-3 In clinical studies, the degrees of lower urinary tract ischemia closely correlated with LUTS severity.4,5 LUTS improvement with alpha-adrenoceptor blockers was associated with significant increase in bladder blood flow.5 In animal models, moderate bladder ischemia has been associated with smooth muscle oxidative damage, muscarinic receptor sensitization, neural overreactivity, and upregulation of excitatory sensory receptors.1-3 Critical aspects of hypersensitivity and smooth muscle instability in bladder ischemia were shown to be mediated by
Financial Disclosure: The authors declare that they have no relevant financial interests. Funding Support: This study was supported by grant BLR&D MERIT 1I01BX001428-01A1 from the U.S. Department of Veterans Affairs. From the Department of Urology, VA Boston Healthcare System, Boston University School of Medicine, Boston, MA Address correspondence to: Kazem M. Azadzoi, M.D., VA Boston Healthcare System, Boston University School of Medicine, Building 1A, Room 315 (151), 150 South Huntington Avenue, Boston, MA 02130. E-mail: [email protected] Submitted: March 17, 2014, accepted (with revisions): July 7, 2014
Published by Elsevier Inc.
nonadrenergic noncholinergic (NANC) mechanisms.1-3 However, the nature of NANC neurotransmission and their corresponding NANC receptors in bladder ischemia has not yet been determined. Studies of human and animal tissue samples suggest that among the NANC system, purinergic nerves and the P2X and P2Y purinoceptors may play leading roles in bladder smooth muscle instability.6-10 In animal models, electrical field stimulation of the bladder was shown to induce acetylcholine and adenosine triphosphate (ATP) corelease from parasympathetic nerve terminals and activate postjunctional muscarinic and purinergic P2X and P2Y receptors.9,10 Purinergic nerves hypersensitivity, differential purinoceptors expression, P2Y-mediated neuronal hyperactivity, and purinoceptor-mediated smooth muscle instability have been documented in dysfunctional bladder samples from human and animal models.10-12 Significant changes in P2X and P2Y receptors expression and reactivity were reported in experimental models of aging bladder,11 interstitial cystitis,12 and outlet obstruction.13 Exposure of cultured cerebrocortical cells to in vitro ischemia resulted in P2X receptors hypersensitivity.14 P2X purinoceptors appeared to mediate ischemiainduced transmission discharge in cultured neural cells.14,15 In the present study, we assessed cystometric and ultrastructural changes of the ischemic bladder, analyzed http://dx.doi.org/10.1016/j.urology.2014.07.023 1249.e1 0090-4295/14
oxidative stress markers, and examined the effects of ischemia on P2X and P2Y purinoceptors expression using a rabbit model.
MATERIALS AND METHODS Animal Model of Chronic Bladder Ischemia Animal care and experimental protocols were in accordance with the guidelines of our institutional animal care and use committee. New Zealand white male rabbits (3-3.5 kg) were anesthetized with continuous inhalation of 1%-2% isoflurane mixed with oxygen. Moderate arterial atherosclerosis and bladder ischemia were produced using bilateral iliac arteries balloon de-endothelialization. Arterial ballooning was repeated 3-4 times on each side while rotating the catheter. The animals received a 0.5%-cholesterol diet for 4 weeks, then a regular rabbit diet. After 8 weeks, changes in the treated animals (n ¼ 10) were compared with age-matched controls (n ¼ 8).
Bladder Blood Flow Measurement and Cystometrogram Hemodynamic measurements and cystometry were performed in both treated and control animals. Under general anesthesia, bladder blood flow was recorded with a laser Doppler flow probe inserted into the bladder wall and connected to a flowmeter. A Foley catheter was placed through the urethra into the bladder. A 23-gauge angiocatheter was inserted into the bladder and connected to a pressure transducer for intravesical pressure recording. The bladder was filled with saline at a rate of 0.8 mL/ min. The bladder volume and intravesical pressure at micturition were recorded. After this, the balloon of the Foley catheter was inflated to prevent leak, the bladder was filled with 25 mL saline and the frequency of spontaneous bladder contractions was recorded.
Transmission Electron Microscopy Bladder tissues were fixed and processed for transmission electron microscopy according to the standard protocols. The following day, the samples were embedded and polymerized. Ultrathin sections were cut, picked up on to copper grids, and stained with lead citrate. Ultrastructure of the ischemic bladder tissues was compared with controls using a JEOL 1200EX microscope (JEOL USA, Inc, Peabody, MA).
Fluorometric Assessment of Oxidative Stress Markers Advanced oxidation protein products (AOPPs) and malondialdehyde (MDA) were analyzed in tissue samples from ischemic and control bladders. AOPPs result from covalent modification of a protein induced directly by reactive oxygen species or indirectly by secondary by-products of oxidative stress. MDA, the product of lipid peroxidation, is a marker of lipid oxidative damage and subsequent deterioration of cell membranes, lipoproteins, and other lipid-containing structures. AOPP assays were performed using OxiSelect AOPP Kit (STA318; Cell Biolabs Inc, San Diego, CA). Samples containing 250 mg/mL protein were prepared, then constant volumes of 200 mL were added to separate wells of a microtiter plate (Cell Biolabs Inc). After adding chloramine reaction initiator and stop solution, the absorbance of each well was recorded by a spectrophotometric plate reader using 340 nm as the primary wavelength. The MDA levels were analyzed by OxiSelect MDA 1249.e2
Adduct ELISA Kit (STA-332; Cell Biolabs, Inc). Samples containing 10 mg/mL protein were prepared, then constant volumes of 100 mL were added to the 96-well protein binding plate, incubated at 37 C for 2 hours. After incubation with antiMDA antibody, samples were washed, incubated with secondary antibodyeHRP conjugate and subsequent color development reagents. The absorbance of each well was read on a microplate reader using 450 nm as the primary wavelength and standardized in micromole as absorbance equivalent.
Western Blotting of Purinoceptors Bladder tissue samples were lysed and centrifuged according to the standard protocols. The supernatant protein concentration was determined by Bradford assay (500-0001; Bio-Rad, Hercules, CA) and diluted in lysis buffer to ensure equal concentration for each sample. After this, equal amounts of proteins were loaded on each lane, run on a 12% SDS-PAGE gel and transferred onto a polyvinylidene fluoride membrane at 250 mA. After blocking, the membranes were incubated with the desired antibodies (Santa Cruz Biotechnology, Inc, Dallas, TX) against P2X1 (sc-25692), P2X2 (sc-12211), P2X3 (sc-25694), P2X4 (sc-28764), P2X5 (sc-25695), P2X6 (sc-166013), P2X7 (sc25698) or the P2Y receptor isoforms at 1:1000 dilution at 4 C for 12 hours. After washing twice, the membranes were incubated with fluorescence-labeled goat antirabbit IgG (1:1000; Invitrogen, Carlsbad, CA) for 2 hours. For tracking molecular weight of the individual protein, the Precision Plus Protein All Blue Standards (Bio-Rad) was included in each SDS-PAGE gel. The optical densities of P2X isoforms were detected and analyzed by Typhoon 8600 (GE Healthcare Life Sciences, Pittsburgh, PA). The relative expression of P2X isoforms was normalized over beta actin optical density values.
Statistical Analysis Data are expressed as mean standard error of the mean. Changes in the treated group were compared with age-matched controls. Significant differences were determined by the t test or the analysis of variance where appropriate followed by post hoc comparisons. Significant differences were determined at P .05 level.
RESULTS Cystometric Changes in Bladder Ischemia Three of the 10 treated animals did not develop significant arterial atherosclerosis and were removed from the study. In the remaining 7 animals, atherosclerotic changes characterized by intimal thickening and significant luminal narrowing of the iliac arteries were present. The hemodynamic significance of arterial atherosclerosis was determined based on bladder blood flow measurements. Arterial atherosclerosis significantly decreased bladder blood flow of the 7 treated animals in comparison with average bladder blood flow in the 8 age-matched control animals (P ¼ .001; Table 1). In cystometry, bladder volume at micturition in bladder ischemia group was significantly smaller than bladder volume at micturition in controls (P ¼ .002; Table 1). The frequency of spontaneous detrusor contractions in bladder ischemia group was significantly greater than that in controls (P ¼ .001; Table 1). Bladder pressure at micturition and intravesical UROLOGY 84 (5), 2014
Table 1. Cystometric changes in ischemic bladders (n ¼ 7) vs age-matched controls (n ¼ 8) Groups Control Ischemia
BBF (mL/min/100 g)
BVmic (mL)
BPmic (mm Hg)
SC/10 min
PRc (mm Hg)
7.5 0.6 3.2 0.2*
24.9 1.2 16.2 0.8*
16.2 2.8 19.5 2.2
1.2 0.3 7.4 0.7*
3.9 1.7 5.5 1.4
BBF, bladder blood flow; BPmic, bladder pressure at micturition; BVmic, bladder volume at micturition; PRc, pressure rise with contraction; SC, spontaneous contractions. * Represents significant changes vs control.
Figure 1. Transmission electron microscopy of microvasculature in tissue samples from the ischemic and age-matched control bladders. Swollen intima with disrupted endothelial cells and marked thickening and fibrosis of subintimal layer are shown in the ischemic bladder tissue vs normal intima with well-defined endothelial cell lining in the control bladder sample. Images are reduced from 18,500.
pressure rise during contractions varied largely among the animals and did not reach significance (Table 1). Ultrastructural Changes in Bladder Ischemia Transmission electron microscopy of ischemic bladder tissues showed significant thickening of epithelial layer, deformation of muscle fascicles, and diffused fibrosis. Marked changes in bladder microvasculature characterized by intimal thickening, diffused subintimal fibrosis and sporadic loss of arteriolar endothelial cells were evident in the ischemic bladder tissues (Fig. 1). Protein Oxidation in Bladder Ischemia The aforementioned structural and cystometric changes in the ischemic bladder were associated with distinct markers of free radical incursion and oxidative damage. Fluorometric analysis of 7 ischemic and 7 control bladder samples revealed significant protein oxidation in tissue samples from the ischemic bladders in comparison with samples from age-matched controls (Fig. 2). This was characterized by significant increase in tissue levels of AOPP in the ischemic bladder tissues (Fig. 2). UROLOGY 84 (5), 2014
Lipid Peroxidation in Bladder Ischemia In addition to protein oxidation, significant increase in lipid peroxidation products was evident in the ischemic bladder tissues. Fluorometric analysis of 7 ischemic and 7 control bladder samples revealed significant increase in malondialdehyde levels, suggesting oxidative damage to lipidcontaining structures including cell membranes (Fig. 2). Changes in P2X Expression Western blotting data from 4 ischemic bladder samples were compared vs 4 controls. P2X1, P2X2, P2X3, P2X4, P2X5, and P2X7 expressions were detected in both ischemic and control rabbit bladder samples (Fig. 3). The P2X6 isoform was not expressed in the rabbit bladder. Structural and functional changes in the chronically ischemic bladder were associated with differential expression of P2X receptor isoforms. Western blotting showed significant upregulation of P2X1, P2X2, P2X3, P2X4, P2X5, and P2X7 in tissue samples from the ischemic bladders in comparison with samples from agematched controls (Fig. 3). P2Y Purinoceptors We examined P2Y1, P2Y2, P2Y4, P2Y6, P2Y7, and P2Y12 expressions in both ischemic and control bladder 1249.e3
Figure 2. Oxidative stress markers in bladder ischemia. The levels of advanced oxidation protein products and malondialdehyde were significantly greater in the ischemic bladder tissues (n ¼ 7) than those of controls (n ¼ 7) suggesting protein oxidation and lipid peroxidation, respectively. An asterisk indicates significant differences vs control. AOPP, advanced oxidation protein products; MDA, malondialdehyde.
tissues. Western blotting did not detect any of the P2Y receptor isoforms in the rabbit bladder.
COMMENT Structural and functional consequences of chronic bladder ischemia depend on the severity and duration of arterial insufficiency.1,2,16 In moderate ischemia, the bladder coordinates a series of molecular responses to promote cell survival and preserve functional integrity of the nerves, smooth muscle cells, and microvasculature. These defensive reactions appear to augment neural reactivity and lead to differential receptor expression, smooth muscle hypersensitivity, and increased contractile activity.1,2,16 In severe ischemia, the bladder undergoes numerous adaptations to cope with lack of perfusion and extreme levels of hypoxia.1,2,16 These adaptations exhaust the bladder energy transduction system due to increased metabolic capacity and lead to mitochondrial damage, smooth muscle atrophy, neurodegeneration, fibrosis, and contractile dysfunction.1,2,16 Factors leading to degeneration of the severely ischemic bladder may relate to nutrient deficiency, severe hypoxia, lack of perfusion to clear metabolic waste, and accumulation of cytotoxic products. In the present study, most of the P2X receptor isoforms, except P2X6, were found in the rabbit bladder. However, antibodies directed against P2Y1, P2Y2, P2Y4, P2Y6, P2Y7, and P2Y12 failed to detect these receptors in the rabbit bladder tissue. Markers of oxidative stress such as protein oxidation and lipid peroxidation were associated with bladder overactivity, ultrastructural damage, and significant increases in P2X1, P2X2, P2X3, P2X4, P2X5, and P2X7 expression. These observations may imply free radical incursion and subsequent upregulation of purinoceptors in response to bladder ischemia. Differential 1249.e4
expression of P2X receptor subtypes may contribute to ischemia-associated bladder noncompliance and increased spontaneous contractions. The bladder purinoceptors localized in smooth muscle and epithelium contribute to important pathophysiological processes including hypersensitivity, pain, and smooth muscle instability.7-14 Increased bladder smooth muscle tone in response to contractile stimuli appeared to involve corelease of cholinergic and purinergic neurotransmission and cross talk mechanisms between the muscarinic and purinergic receptors.7-9,11 It was shown that muscarinic receptor antagonists fail to fully inhibit nerve-stimulated bladder smooth muscle contractions.6-9,17 Most of the remaining contractions after antimuscarinic treatment were inhibited by the P2X receptor antagonists. These observations suggest purinergic regulation of antimuscarinic-resistant smooth muscle contractility and imply therapeutic implication of P2X purinoceptors in antimuscarinic-resistant bladder contractions. This concept is supported by studies of human bladder tissues showing greater involvement of P2X receptors in samples from symptomatic elderly patients vs samples from younger asymptomatic controls.6,11 In human bladder, P2X3 receptors have been localized on the urothelium, whereas P2X2 receptors were found in smooth muscle cells.18 P2X3 receptors have been implicated in neuralgia and believed to mediate hyporeflexia. The precise role of P2X3 in human bladder has not been thoroughly investigated.18,19 In experimental models, P2X3 knockout mice exhibited normal bladder pressure, lower voiding frequency, increased bladder capacity, and larger voiding volumes.19 Large numbers of homomeric and mixed subtype clusters of P2X1, P2X2, P2X3, P2X4, P2X5, and P2X6 receptors have been localized in the rat bladder smooth muscle and epithelium.20 P2X1 receptor immunoreactivity has been detected mainly on the bladder smooth muscle membranes, whereas P2X2, P2X5, and P2X6 immunoreactivity were found throughout the UROLOGY 84 (5), 2014
Figure 3. P2X purinoceptor expressions in chronic rabbit bladder ischemia. This figure shows significant increases in P2X1, P2X2, P2X3, P2X4, P2X5, and P2X7 protein expressions in the ischemic bladder tissues (n ¼ 4) in comparison with those of controls (n ¼ 4). An asterisk indicates significant differences in the ischemic bladders vs age-matched controls. OD, optical density.
muscular layer.20 The role of purinoceptors in bladder hyporeflexia and pain was documented in mice.21 These studies revealed broadly expressed bladder purinoceptors that appeared to be modified and differently distributed under the disease conditions.18-20 Differential expression of P2X receptors may provoke a spectrum of pathologic processes and lead to functional consequences. P2X receptors mediate important aspects of intracellular ATP signaling involved in intracellular calcium levels, phospholipase activity, vascular smooth muscle tone, neurotransmission discharge, and increased contractile activity.22 Functional changes in the P2X2 knockout mice and P2X2/P2X3 double knockout mice provided valuable information on the role of purinoceptors in neurogenic bladder contractions.22 Significant decrease in sensory neuronal activity was recorded in the UROLOGY 84 (5), 2014
P2X2 knockout mice, whereas reduced pain-related behavior, decreased bladder reflexes, and diminished pelvic afferent neural activity were found in the P2X2/ P2X3 double knockout mice.22 Excitation of sensory neurons in human bladder resulted in ATP release and subsequent signaling via P2X purinoceptors. This is thought to provoke pain, hypersensitivity, and smooth muscle contraction.22 Alterations of P2X receptor isoforms in chronic bladder ischemia may have important clinical implications. Arterial insufficiency may be an independent agingassociated sex-independent factor in the development of nonneurogenic, nonobstructed overactive bladder. Clinical and basic research data suggest the role of ischemia in a range of pathophysiological episodes including bladder hypersensitivity, detrusor instability, 1249.e5
fibrosis, and noncompliance.1-5,16,23,24 Oxidative stress, a noxious manifestation of ischemia, results from unchecked formation of free radicals, antioxidant deficiency, and accumulation of metabolic waste. Free radical incursion of surrounding structures activate a number of downstream pathways and lead to oxidative damage to receptors, nerve fibers, and smooth muscle cells.1,16,23 In a previous study, we found that human bladder smooth muscle cells exposure to hypoxia and oxidative stress resulted in critical subcellular changes involving cell membrane, mitochondria, endoplasmic reticulum, and lysosomes.23 These changes were consistent with cellular and molecular alterations in chronic rabbit bladder ischemia.1,16 It has been shown that P2X purinoceptors are primary targets of free radical incursion and also important mediators of free radical formation.25 Stimulation of P2X receptors in blood vessels increased superoxide production and led to hypersensitivity and smooth muscle cell contractility.25 Extracellular ATP was shown to stimulate free radical production in airway epithelial cells by a mechanism involving nicotinamide adenine dinucleotide phosphate oxidase.26 These observations suggest that ATP and P2X receptors not only react to redox conditions but also trigger free radical production and contribute to redox reactions.25,26 Coordinated P2X reactions and discrepancy in atropine sensitivity of dysfunctional bladder vs normal suggest that purinoceptor changes in disordered conditions may depend on the pathologic state.26 Studies of bladder samples from patients with idiopathic detrusor overactivity have shown that purinergic transmission and P2X receptors contribute to almost 50% of pathologic contractions.26 Aging-related detrusor overactivity has been associated with P2X hypersensitivity and increased contractile response to ATP, suggesting aging-associated increase in purinergic activity.27 Upregulation of P2X2 and downregulation of P2X3 and P2X5 in idiopathic detrusor instability and increased P2X3 expression in neurogenic bladder have also been documented.26,28,29 Decreased protein levels of all P2X isoforms were documented in detrusor and subepithelial layer of samples from patients with sensory urgency.30 These observations in human and animal tissues imply explicit responses of P2X receptors to distinct features of dysfunctional bladder such as outlet obstruction, overactivity, sensory urgency, interstitial cystitis, and arterial insufficiency. The precise mechanisms by which purinergic activity emerges and P2X receptors intervene in pathologic bladder conditions remain unknown. It is thought that in addition to P2X modifications, incomplete breakdown of ATP by ATPases and subsequent ATP accumulation under the disease conditions could also contribute to increased bladder contractility.27 Precise localization of P2X receptor isoforms in bladder layers may provide useful diagnostic markers and could lead to newer therapeutic targets. In view of increased purinergic signaling in bladder conditions involving overactivity and pain, inhibitors of P2X receptor isoforms may have important 1249.e6
prophylactic and therapeutic implications. Therapeutic strategies targeting disordered P2X receptor isoforms may help counteract bladder symptoms and could lead to new strategies against detrusor overactivity and LUTS.
CONCLUSION Upregulation of P2X purinergic receptor isoforms in bladder ischemia was associated with markers of oxidative stress, detrusor overactivity, and ultrastructural damage. P2X receptors, potential targets of oxidative radicals, could also mediate free radical formation and contribute to oxidative damage in the bladder. Differential P2X expression may be a disease-specific phenomenon in critical aspects of bladder dysfunction including overactivity, hypersensitivity, and pain. Despite the documented evidence, however, mechanistic principles of purinergic transmission and purinoceptor expression in ischemia and their involvement in bladder dysfunction remain unclear and warrant future investigation. References 1. Azadzoi KM, Tarcan T, Kozlowski R, et al. Overactivity and structural changes in the chronically ischemic bladder. J Urol. 1999; 162:1768-1778. 2. Azadzoi KM, Radisavljevic ZM, Golabek T, et al. Oxidative modification of mitochondrial integrity and nerve fiber density in the ischemic overactive bladder. J Urol. 2010;183:362-369. 3. Nomiya M, Sagawa K, Yazaki J, et al. Increased bladder activity is associated with elevated oxidative stress markers and proinflammatory cytokines in a rat model of atherosclerosis-induced chronic bladder ischemia. Neurourol Urodyn. 2012;31:185-189. 4. Pinggera GM, Michael M, Eberhard S, et al. Association of lower urinary tract symptoms and chronic ischaemia of the lower urinary tract in elderly women and men: assessment using colour Doppler ultrasonography. Br J Urol. 2008;102:470-474. 5. Pinggera GM, Mitterberger M, Pallwein L, et al. Alpha-blockers improve chronic ischaemia of the lower urinary tract in patients with lower urinary tract symptoms. BJU Int. 2007;101:319-324. 6. Tagliani M, Candura SM, Di Nucci A, et al. A re-appraisal of the nature of the atropine-resistant contraction to electrical field stimulation in the human isolated detrusor muscle. Naunyn Schmiedebergs Arch Pharmacol. 1997;356:750-755. 7. McMurray G, Dass N, Brading AF, et al. Purinoceptor subtypes mediating contraction and relaxation of marmoset urinary bladder smooth muscle. Br J Pharmacol. 1998;123:1579-1586. 8. Barajas-Lopez C, Espinosa-Luna R, Zhu Y, et al. Functional interactions between nicotinic and P2X channels in short-term cultures of guinea-pig submucosal neurons. J Physiol. 1998;513(Pt 3): 671-683. 9. Somogyi GT, Zernova GV, Yoshiyama M, et al. Frequency dependence of muscarinic facilitation of transmitter release in urinary bladder strips from neurally intact or chronic spinal cord transected rats. Br J Pharmacol. 1998;125:241-246. 10. Chen X, Molliver DC, Gebhart GF. The P2Y2 receptor sensitizes mouse bladder sensory neurons and facilitates purinergic currents. J Neurosci. 2010;30:2365-2372. 11. Yoshida M, Homma Y, Inadome A, et al. Age-related changes in cholinergic and purinergic neurotransmission in human isolated bladder smooth muscles. Exp Gerontol. 2001;36:99-109. 12. Birder LA, Ruan HZ, Chopra B, et al. Alterations in P2X and P2Y purinergic receptor expression in urinary bladder from normal cats and cats with interstitial cystitis. Am J Physiol Renal Physiol. 2004; 287:F1084-F1091.
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13. Kim JC, Yoo JS, Park EY, et al. Muscarinic and purinergic receptor expression in the urothelium of rats with detrusor overactivity induced by bladder outlet obstruction. BJU Int. 2008;101:371-375. 14. Wirkner K, K€ ofalvi A, Fischer W, et al. Supersensitivity of P2X receptors in cerebrocortical cell cultures after in vitro ischemia. J Neurochem. 2005;95:1421-1437. 15. Milano PM, Douillet CD, Riesenman PJ, et al. Intestinal ischemiareperfusion injury alters purinergic receptor expression in clinically relevant extraintestinal organs. J Surg Res. 2008;145:272-278. 16. Azadzoi KM, Chen B, Radisavljevic Z, Mike Siroky. Molecular reactions and ultrastructural damage in the chronically ischemic bladder. J Urol. 2011;186:2115-2122. 17. Kennedy C, Tasker PN, Gallacher G, et al. Identification of atropine- and P2X1 receptor antagonist-resistant, neurogenic contractions of the urinary bladder. J Neurosci. 2007;27:845-851. 18. Elneil S, Skepper JN, Kidd EJ, et al. Distribution of P2X(1) and P2X(3) receptors in the rat and human urinary bladder. Pharmacology. 2001;63:120-128. 19. Cockayne DA, Hamilton SG, Zhu QM, et al. Urinary bladder hyporeflexia and reduced pain-related behaviour in P2X3-deficient mice. Nature. 2000;407:1011-1015. 20. Lee HY, Bardini M, Burnstock G. Distribution of P2X receptors in the urinary bladder and the ureter of the rat. J Urol. 2000;163:20022007. 21. Ferguson DR, Kennedy I, Burton TJ. ATP is released from rabbit urinary bladder epithelial cells by hydrostatic pressure changesea possible sensory mechanism? J Physiol. 1997;505:503-511.
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22. Cockayne DA, Dunn PM, Zhong Y, et al. P2X2 knockout mice and P2X2/P2X3 double knockout mice reveal a role for the P2X2 receptor subunit in mediating multiple sensory effects of ATP. J Physiol. 2005;567:621-639. 23. Azadzoi KM, Yalla SV, Siroky MB. Human bladder smooth muscle damage in disturbed oxygen tension. Urology. 2011;78:967.e9-967. e15. 24. Kershen RT, Azadzoi KA, Siroky MB. Blood flow, pressure and compliance in the male human bladder. J Urol. 2002;168:121-125. 25. Judkins CP, Sobey CG, Dang TT, et al. NADPH-induced contractions of mouse aorta do not involve NADPH oxidase: a role for P2X receptors. J Pharmacol Exp Ther. 2006;317:644-650. 26. O’Reilly BA, Kosaka AH, Knight GF, et al. P2X receptors and their role in female idiopathic detrusor instability. J Urol. 2002;167: 157-164. 27. Yoshida M, Miyamae K, Iwashita H, et al. Management of detrusor dysfunction in the elderly: changes in acetylcholine and adenosine triphosphate release during aging. Urology. 2004;63:17-23. 28. Moore KH, Ray FR, Barden JA. Loss of purinergic P2X(3) and P2X(5) receptors innervation in human detrusor from adults with urge incontinence. J Neurosci. 2001;21:RCS166. 29. Brady CM, Apostolidis A, Yiangou Y, et al. P2X3-immunoreactive nerve fibres in neurogenic detrusor overactivity and the effect of intravesical resiniferatoxin. Eur Urol. 2004;46:247-253. 30. Ray FR, Moore KH, Hansen MA, et al. Loss of purinergic P2X receptor innervation in human detrusor and subepithelium from adults with sensory urgency. Cell Tissue Res. 2003;314:351-359.
1249.e7
MATERIALS AND METHODS
RESULTS
CONCLUSION
To study the effects of chronic ischemia on bladder purinoceptors. A close correlation between bladder ischemia and lower urinary tract symptoms has been reported. Purinoceptors contribute to important aspects of bladder function including sensation, neural signaling, and voiding contraction. Our goal was to examine purinoceptors expression in the ischemic overactive bladder. Moderate bladder ischemia was produced in rabbits by creating bilateral iliac artery atherosclerosis. After 8 weeks, bladder blood flow was measured, and cystometrograms were obtained. Bladder tissues from 8-week ischemic and age-matched control bladders were processed for the analysis of oxidative stress markers, P2X and P2Y purinoceptors expression, and transmission electron microscopy. Arterial atherosclerosis significantly decreased bladder blood flow. Markers of oxidative stress characterized by increased levels of advanced oxidation protein products and malondialdehyde were evident in the ischemic bladder tissues. Chronic ischemia and oxidative stress decreased the bladder capacity and increased spontaneous bladder contractions. Bladder pressure at micturition and intravesical pressure rise during contractions tended to be greater in the ischemic bladder but did not reach significance. Transmission electron microscopy showed smooth muscle cell and microvasculature structural damage and diffuse fibrosis. These changes in the ischemic bladder were associated with significant increases in purinoceptors P2X1, P2X2, P2X3, P2X4, P2X5, and P2X7 expression. The P2Y isoforms were not expressed in the rabbit bladder. Structural and functional changes in the chronically ischemic bladder were associated with upregulation of P2X receptor isoforms. Increased P2X expression may play a role in ischemia-induced bladder overactivity and noncompliance. UROLOGY 84: 1249.e1e1249.e7, 2014. Published by Elsevier Inc.
P
elvic arterial insufficiency may contribute to agingassociated bladder dysfunction and lower urinary tract symptoms (LUTS).1-5 In experimental models, moderate ischemia resulted in bladder overactivity, whereas severe ischemia diminished smooth muscle contractile responses.1-3 In clinical studies, the degrees of lower urinary tract ischemia closely correlated with LUTS severity.4,5 LUTS improvement with alpha-adrenoceptor blockers was associated with significant increase in bladder blood flow.5 In animal models, moderate bladder ischemia has been associated with smooth muscle oxidative damage, muscarinic receptor sensitization, neural overreactivity, and upregulation of excitatory sensory receptors.1-3 Critical aspects of hypersensitivity and smooth muscle instability in bladder ischemia were shown to be mediated by
Financial Disclosure: The authors declare that they have no relevant financial interests. Funding Support: This study was supported by grant BLR&D MERIT 1I01BX001428-01A1 from the U.S. Department of Veterans Affairs. From the Department of Urology, VA Boston Healthcare System, Boston University School of Medicine, Boston, MA Address correspondence to: Kazem M. Azadzoi, M.D., VA Boston Healthcare System, Boston University School of Medicine, Building 1A, Room 315 (151), 150 South Huntington Avenue, Boston, MA 02130. E-mail: [email protected] Submitted: March 17, 2014, accepted (with revisions): July 7, 2014
Published by Elsevier Inc.
nonadrenergic noncholinergic (NANC) mechanisms.1-3 However, the nature of NANC neurotransmission and their corresponding NANC receptors in bladder ischemia has not yet been determined. Studies of human and animal tissue samples suggest that among the NANC system, purinergic nerves and the P2X and P2Y purinoceptors may play leading roles in bladder smooth muscle instability.6-10 In animal models, electrical field stimulation of the bladder was shown to induce acetylcholine and adenosine triphosphate (ATP) corelease from parasympathetic nerve terminals and activate postjunctional muscarinic and purinergic P2X and P2Y receptors.9,10 Purinergic nerves hypersensitivity, differential purinoceptors expression, P2Y-mediated neuronal hyperactivity, and purinoceptor-mediated smooth muscle instability have been documented in dysfunctional bladder samples from human and animal models.10-12 Significant changes in P2X and P2Y receptors expression and reactivity were reported in experimental models of aging bladder,11 interstitial cystitis,12 and outlet obstruction.13 Exposure of cultured cerebrocortical cells to in vitro ischemia resulted in P2X receptors hypersensitivity.14 P2X purinoceptors appeared to mediate ischemiainduced transmission discharge in cultured neural cells.14,15 In the present study, we assessed cystometric and ultrastructural changes of the ischemic bladder, analyzed http://dx.doi.org/10.1016/j.urology.2014.07.023 1249.e1 0090-4295/14
oxidative stress markers, and examined the effects of ischemia on P2X and P2Y purinoceptors expression using a rabbit model.
MATERIALS AND METHODS Animal Model of Chronic Bladder Ischemia Animal care and experimental protocols were in accordance with the guidelines of our institutional animal care and use committee. New Zealand white male rabbits (3-3.5 kg) were anesthetized with continuous inhalation of 1%-2% isoflurane mixed with oxygen. Moderate arterial atherosclerosis and bladder ischemia were produced using bilateral iliac arteries balloon de-endothelialization. Arterial ballooning was repeated 3-4 times on each side while rotating the catheter. The animals received a 0.5%-cholesterol diet for 4 weeks, then a regular rabbit diet. After 8 weeks, changes in the treated animals (n ¼ 10) were compared with age-matched controls (n ¼ 8).
Bladder Blood Flow Measurement and Cystometrogram Hemodynamic measurements and cystometry were performed in both treated and control animals. Under general anesthesia, bladder blood flow was recorded with a laser Doppler flow probe inserted into the bladder wall and connected to a flowmeter. A Foley catheter was placed through the urethra into the bladder. A 23-gauge angiocatheter was inserted into the bladder and connected to a pressure transducer for intravesical pressure recording. The bladder was filled with saline at a rate of 0.8 mL/ min. The bladder volume and intravesical pressure at micturition were recorded. After this, the balloon of the Foley catheter was inflated to prevent leak, the bladder was filled with 25 mL saline and the frequency of spontaneous bladder contractions was recorded.
Transmission Electron Microscopy Bladder tissues were fixed and processed for transmission electron microscopy according to the standard protocols. The following day, the samples were embedded and polymerized. Ultrathin sections were cut, picked up on to copper grids, and stained with lead citrate. Ultrastructure of the ischemic bladder tissues was compared with controls using a JEOL 1200EX microscope (JEOL USA, Inc, Peabody, MA).
Fluorometric Assessment of Oxidative Stress Markers Advanced oxidation protein products (AOPPs) and malondialdehyde (MDA) were analyzed in tissue samples from ischemic and control bladders. AOPPs result from covalent modification of a protein induced directly by reactive oxygen species or indirectly by secondary by-products of oxidative stress. MDA, the product of lipid peroxidation, is a marker of lipid oxidative damage and subsequent deterioration of cell membranes, lipoproteins, and other lipid-containing structures. AOPP assays were performed using OxiSelect AOPP Kit (STA318; Cell Biolabs Inc, San Diego, CA). Samples containing 250 mg/mL protein were prepared, then constant volumes of 200 mL were added to separate wells of a microtiter plate (Cell Biolabs Inc). After adding chloramine reaction initiator and stop solution, the absorbance of each well was recorded by a spectrophotometric plate reader using 340 nm as the primary wavelength. The MDA levels were analyzed by OxiSelect MDA 1249.e2
Adduct ELISA Kit (STA-332; Cell Biolabs, Inc). Samples containing 10 mg/mL protein were prepared, then constant volumes of 100 mL were added to the 96-well protein binding plate, incubated at 37 C for 2 hours. After incubation with antiMDA antibody, samples were washed, incubated with secondary antibodyeHRP conjugate and subsequent color development reagents. The absorbance of each well was read on a microplate reader using 450 nm as the primary wavelength and standardized in micromole as absorbance equivalent.
Western Blotting of Purinoceptors Bladder tissue samples were lysed and centrifuged according to the standard protocols. The supernatant protein concentration was determined by Bradford assay (500-0001; Bio-Rad, Hercules, CA) and diluted in lysis buffer to ensure equal concentration for each sample. After this, equal amounts of proteins were loaded on each lane, run on a 12% SDS-PAGE gel and transferred onto a polyvinylidene fluoride membrane at 250 mA. After blocking, the membranes were incubated with the desired antibodies (Santa Cruz Biotechnology, Inc, Dallas, TX) against P2X1 (sc-25692), P2X2 (sc-12211), P2X3 (sc-25694), P2X4 (sc-28764), P2X5 (sc-25695), P2X6 (sc-166013), P2X7 (sc25698) or the P2Y receptor isoforms at 1:1000 dilution at 4 C for 12 hours. After washing twice, the membranes were incubated with fluorescence-labeled goat antirabbit IgG (1:1000; Invitrogen, Carlsbad, CA) for 2 hours. For tracking molecular weight of the individual protein, the Precision Plus Protein All Blue Standards (Bio-Rad) was included in each SDS-PAGE gel. The optical densities of P2X isoforms were detected and analyzed by Typhoon 8600 (GE Healthcare Life Sciences, Pittsburgh, PA). The relative expression of P2X isoforms was normalized over beta actin optical density values.
Statistical Analysis Data are expressed as mean standard error of the mean. Changes in the treated group were compared with age-matched controls. Significant differences were determined by the t test or the analysis of variance where appropriate followed by post hoc comparisons. Significant differences were determined at P .05 level.
RESULTS Cystometric Changes in Bladder Ischemia Three of the 10 treated animals did not develop significant arterial atherosclerosis and were removed from the study. In the remaining 7 animals, atherosclerotic changes characterized by intimal thickening and significant luminal narrowing of the iliac arteries were present. The hemodynamic significance of arterial atherosclerosis was determined based on bladder blood flow measurements. Arterial atherosclerosis significantly decreased bladder blood flow of the 7 treated animals in comparison with average bladder blood flow in the 8 age-matched control animals (P ¼ .001; Table 1). In cystometry, bladder volume at micturition in bladder ischemia group was significantly smaller than bladder volume at micturition in controls (P ¼ .002; Table 1). The frequency of spontaneous detrusor contractions in bladder ischemia group was significantly greater than that in controls (P ¼ .001; Table 1). Bladder pressure at micturition and intravesical UROLOGY 84 (5), 2014
Table 1. Cystometric changes in ischemic bladders (n ¼ 7) vs age-matched controls (n ¼ 8) Groups Control Ischemia
BBF (mL/min/100 g)
BVmic (mL)
BPmic (mm Hg)
SC/10 min
PRc (mm Hg)
7.5 0.6 3.2 0.2*
24.9 1.2 16.2 0.8*
16.2 2.8 19.5 2.2
1.2 0.3 7.4 0.7*
3.9 1.7 5.5 1.4
BBF, bladder blood flow; BPmic, bladder pressure at micturition; BVmic, bladder volume at micturition; PRc, pressure rise with contraction; SC, spontaneous contractions. * Represents significant changes vs control.
Figure 1. Transmission electron microscopy of microvasculature in tissue samples from the ischemic and age-matched control bladders. Swollen intima with disrupted endothelial cells and marked thickening and fibrosis of subintimal layer are shown in the ischemic bladder tissue vs normal intima with well-defined endothelial cell lining in the control bladder sample. Images are reduced from 18,500.
pressure rise during contractions varied largely among the animals and did not reach significance (Table 1). Ultrastructural Changes in Bladder Ischemia Transmission electron microscopy of ischemic bladder tissues showed significant thickening of epithelial layer, deformation of muscle fascicles, and diffused fibrosis. Marked changes in bladder microvasculature characterized by intimal thickening, diffused subintimal fibrosis and sporadic loss of arteriolar endothelial cells were evident in the ischemic bladder tissues (Fig. 1). Protein Oxidation in Bladder Ischemia The aforementioned structural and cystometric changes in the ischemic bladder were associated with distinct markers of free radical incursion and oxidative damage. Fluorometric analysis of 7 ischemic and 7 control bladder samples revealed significant protein oxidation in tissue samples from the ischemic bladders in comparison with samples from age-matched controls (Fig. 2). This was characterized by significant increase in tissue levels of AOPP in the ischemic bladder tissues (Fig. 2). UROLOGY 84 (5), 2014
Lipid Peroxidation in Bladder Ischemia In addition to protein oxidation, significant increase in lipid peroxidation products was evident in the ischemic bladder tissues. Fluorometric analysis of 7 ischemic and 7 control bladder samples revealed significant increase in malondialdehyde levels, suggesting oxidative damage to lipidcontaining structures including cell membranes (Fig. 2). Changes in P2X Expression Western blotting data from 4 ischemic bladder samples were compared vs 4 controls. P2X1, P2X2, P2X3, P2X4, P2X5, and P2X7 expressions were detected in both ischemic and control rabbit bladder samples (Fig. 3). The P2X6 isoform was not expressed in the rabbit bladder. Structural and functional changes in the chronically ischemic bladder were associated with differential expression of P2X receptor isoforms. Western blotting showed significant upregulation of P2X1, P2X2, P2X3, P2X4, P2X5, and P2X7 in tissue samples from the ischemic bladders in comparison with samples from agematched controls (Fig. 3). P2Y Purinoceptors We examined P2Y1, P2Y2, P2Y4, P2Y6, P2Y7, and P2Y12 expressions in both ischemic and control bladder 1249.e3
Figure 2. Oxidative stress markers in bladder ischemia. The levels of advanced oxidation protein products and malondialdehyde were significantly greater in the ischemic bladder tissues (n ¼ 7) than those of controls (n ¼ 7) suggesting protein oxidation and lipid peroxidation, respectively. An asterisk indicates significant differences vs control. AOPP, advanced oxidation protein products; MDA, malondialdehyde.
tissues. Western blotting did not detect any of the P2Y receptor isoforms in the rabbit bladder.
COMMENT Structural and functional consequences of chronic bladder ischemia depend on the severity and duration of arterial insufficiency.1,2,16 In moderate ischemia, the bladder coordinates a series of molecular responses to promote cell survival and preserve functional integrity of the nerves, smooth muscle cells, and microvasculature. These defensive reactions appear to augment neural reactivity and lead to differential receptor expression, smooth muscle hypersensitivity, and increased contractile activity.1,2,16 In severe ischemia, the bladder undergoes numerous adaptations to cope with lack of perfusion and extreme levels of hypoxia.1,2,16 These adaptations exhaust the bladder energy transduction system due to increased metabolic capacity and lead to mitochondrial damage, smooth muscle atrophy, neurodegeneration, fibrosis, and contractile dysfunction.1,2,16 Factors leading to degeneration of the severely ischemic bladder may relate to nutrient deficiency, severe hypoxia, lack of perfusion to clear metabolic waste, and accumulation of cytotoxic products. In the present study, most of the P2X receptor isoforms, except P2X6, were found in the rabbit bladder. However, antibodies directed against P2Y1, P2Y2, P2Y4, P2Y6, P2Y7, and P2Y12 failed to detect these receptors in the rabbit bladder tissue. Markers of oxidative stress such as protein oxidation and lipid peroxidation were associated with bladder overactivity, ultrastructural damage, and significant increases in P2X1, P2X2, P2X3, P2X4, P2X5, and P2X7 expression. These observations may imply free radical incursion and subsequent upregulation of purinoceptors in response to bladder ischemia. Differential 1249.e4
expression of P2X receptor subtypes may contribute to ischemia-associated bladder noncompliance and increased spontaneous contractions. The bladder purinoceptors localized in smooth muscle and epithelium contribute to important pathophysiological processes including hypersensitivity, pain, and smooth muscle instability.7-14 Increased bladder smooth muscle tone in response to contractile stimuli appeared to involve corelease of cholinergic and purinergic neurotransmission and cross talk mechanisms between the muscarinic and purinergic receptors.7-9,11 It was shown that muscarinic receptor antagonists fail to fully inhibit nerve-stimulated bladder smooth muscle contractions.6-9,17 Most of the remaining contractions after antimuscarinic treatment were inhibited by the P2X receptor antagonists. These observations suggest purinergic regulation of antimuscarinic-resistant smooth muscle contractility and imply therapeutic implication of P2X purinoceptors in antimuscarinic-resistant bladder contractions. This concept is supported by studies of human bladder tissues showing greater involvement of P2X receptors in samples from symptomatic elderly patients vs samples from younger asymptomatic controls.6,11 In human bladder, P2X3 receptors have been localized on the urothelium, whereas P2X2 receptors were found in smooth muscle cells.18 P2X3 receptors have been implicated in neuralgia and believed to mediate hyporeflexia. The precise role of P2X3 in human bladder has not been thoroughly investigated.18,19 In experimental models, P2X3 knockout mice exhibited normal bladder pressure, lower voiding frequency, increased bladder capacity, and larger voiding volumes.19 Large numbers of homomeric and mixed subtype clusters of P2X1, P2X2, P2X3, P2X4, P2X5, and P2X6 receptors have been localized in the rat bladder smooth muscle and epithelium.20 P2X1 receptor immunoreactivity has been detected mainly on the bladder smooth muscle membranes, whereas P2X2, P2X5, and P2X6 immunoreactivity were found throughout the UROLOGY 84 (5), 2014
Figure 3. P2X purinoceptor expressions in chronic rabbit bladder ischemia. This figure shows significant increases in P2X1, P2X2, P2X3, P2X4, P2X5, and P2X7 protein expressions in the ischemic bladder tissues (n ¼ 4) in comparison with those of controls (n ¼ 4). An asterisk indicates significant differences in the ischemic bladders vs age-matched controls. OD, optical density.
muscular layer.20 The role of purinoceptors in bladder hyporeflexia and pain was documented in mice.21 These studies revealed broadly expressed bladder purinoceptors that appeared to be modified and differently distributed under the disease conditions.18-20 Differential expression of P2X receptors may provoke a spectrum of pathologic processes and lead to functional consequences. P2X receptors mediate important aspects of intracellular ATP signaling involved in intracellular calcium levels, phospholipase activity, vascular smooth muscle tone, neurotransmission discharge, and increased contractile activity.22 Functional changes in the P2X2 knockout mice and P2X2/P2X3 double knockout mice provided valuable information on the role of purinoceptors in neurogenic bladder contractions.22 Significant decrease in sensory neuronal activity was recorded in the UROLOGY 84 (5), 2014
P2X2 knockout mice, whereas reduced pain-related behavior, decreased bladder reflexes, and diminished pelvic afferent neural activity were found in the P2X2/ P2X3 double knockout mice.22 Excitation of sensory neurons in human bladder resulted in ATP release and subsequent signaling via P2X purinoceptors. This is thought to provoke pain, hypersensitivity, and smooth muscle contraction.22 Alterations of P2X receptor isoforms in chronic bladder ischemia may have important clinical implications. Arterial insufficiency may be an independent agingassociated sex-independent factor in the development of nonneurogenic, nonobstructed overactive bladder. Clinical and basic research data suggest the role of ischemia in a range of pathophysiological episodes including bladder hypersensitivity, detrusor instability, 1249.e5
fibrosis, and noncompliance.1-5,16,23,24 Oxidative stress, a noxious manifestation of ischemia, results from unchecked formation of free radicals, antioxidant deficiency, and accumulation of metabolic waste. Free radical incursion of surrounding structures activate a number of downstream pathways and lead to oxidative damage to receptors, nerve fibers, and smooth muscle cells.1,16,23 In a previous study, we found that human bladder smooth muscle cells exposure to hypoxia and oxidative stress resulted in critical subcellular changes involving cell membrane, mitochondria, endoplasmic reticulum, and lysosomes.23 These changes were consistent with cellular and molecular alterations in chronic rabbit bladder ischemia.1,16 It has been shown that P2X purinoceptors are primary targets of free radical incursion and also important mediators of free radical formation.25 Stimulation of P2X receptors in blood vessels increased superoxide production and led to hypersensitivity and smooth muscle cell contractility.25 Extracellular ATP was shown to stimulate free radical production in airway epithelial cells by a mechanism involving nicotinamide adenine dinucleotide phosphate oxidase.26 These observations suggest that ATP and P2X receptors not only react to redox conditions but also trigger free radical production and contribute to redox reactions.25,26 Coordinated P2X reactions and discrepancy in atropine sensitivity of dysfunctional bladder vs normal suggest that purinoceptor changes in disordered conditions may depend on the pathologic state.26 Studies of bladder samples from patients with idiopathic detrusor overactivity have shown that purinergic transmission and P2X receptors contribute to almost 50% of pathologic contractions.26 Aging-related detrusor overactivity has been associated with P2X hypersensitivity and increased contractile response to ATP, suggesting aging-associated increase in purinergic activity.27 Upregulation of P2X2 and downregulation of P2X3 and P2X5 in idiopathic detrusor instability and increased P2X3 expression in neurogenic bladder have also been documented.26,28,29 Decreased protein levels of all P2X isoforms were documented in detrusor and subepithelial layer of samples from patients with sensory urgency.30 These observations in human and animal tissues imply explicit responses of P2X receptors to distinct features of dysfunctional bladder such as outlet obstruction, overactivity, sensory urgency, interstitial cystitis, and arterial insufficiency. The precise mechanisms by which purinergic activity emerges and P2X receptors intervene in pathologic bladder conditions remain unknown. It is thought that in addition to P2X modifications, incomplete breakdown of ATP by ATPases and subsequent ATP accumulation under the disease conditions could also contribute to increased bladder contractility.27 Precise localization of P2X receptor isoforms in bladder layers may provide useful diagnostic markers and could lead to newer therapeutic targets. In view of increased purinergic signaling in bladder conditions involving overactivity and pain, inhibitors of P2X receptor isoforms may have important 1249.e6
prophylactic and therapeutic implications. Therapeutic strategies targeting disordered P2X receptor isoforms may help counteract bladder symptoms and could lead to new strategies against detrusor overactivity and LUTS.
CONCLUSION Upregulation of P2X purinergic receptor isoforms in bladder ischemia was associated with markers of oxidative stress, detrusor overactivity, and ultrastructural damage. P2X receptors, potential targets of oxidative radicals, could also mediate free radical formation and contribute to oxidative damage in the bladder. Differential P2X expression may be a disease-specific phenomenon in critical aspects of bladder dysfunction including overactivity, hypersensitivity, and pain. Despite the documented evidence, however, mechanistic principles of purinergic transmission and purinoceptor expression in ischemia and their involvement in bladder dysfunction remain unclear and warrant future investigation. References 1. Azadzoi KM, Tarcan T, Kozlowski R, et al. Overactivity and structural changes in the chronically ischemic bladder. J Urol. 1999; 162:1768-1778. 2. Azadzoi KM, Radisavljevic ZM, Golabek T, et al. Oxidative modification of mitochondrial integrity and nerve fiber density in the ischemic overactive bladder. J Urol. 2010;183:362-369. 3. Nomiya M, Sagawa K, Yazaki J, et al. Increased bladder activity is associated with elevated oxidative stress markers and proinflammatory cytokines in a rat model of atherosclerosis-induced chronic bladder ischemia. Neurourol Urodyn. 2012;31:185-189. 4. Pinggera GM, Michael M, Eberhard S, et al. Association of lower urinary tract symptoms and chronic ischaemia of the lower urinary tract in elderly women and men: assessment using colour Doppler ultrasonography. Br J Urol. 2008;102:470-474. 5. Pinggera GM, Mitterberger M, Pallwein L, et al. Alpha-blockers improve chronic ischaemia of the lower urinary tract in patients with lower urinary tract symptoms. BJU Int. 2007;101:319-324. 6. Tagliani M, Candura SM, Di Nucci A, et al. A re-appraisal of the nature of the atropine-resistant contraction to electrical field stimulation in the human isolated detrusor muscle. Naunyn Schmiedebergs Arch Pharmacol. 1997;356:750-755. 7. McMurray G, Dass N, Brading AF, et al. Purinoceptor subtypes mediating contraction and relaxation of marmoset urinary bladder smooth muscle. Br J Pharmacol. 1998;123:1579-1586. 8. Barajas-Lopez C, Espinosa-Luna R, Zhu Y, et al. Functional interactions between nicotinic and P2X channels in short-term cultures of guinea-pig submucosal neurons. J Physiol. 1998;513(Pt 3): 671-683. 9. Somogyi GT, Zernova GV, Yoshiyama M, et al. Frequency dependence of muscarinic facilitation of transmitter release in urinary bladder strips from neurally intact or chronic spinal cord transected rats. Br J Pharmacol. 1998;125:241-246. 10. Chen X, Molliver DC, Gebhart GF. The P2Y2 receptor sensitizes mouse bladder sensory neurons and facilitates purinergic currents. J Neurosci. 2010;30:2365-2372. 11. Yoshida M, Homma Y, Inadome A, et al. Age-related changes in cholinergic and purinergic neurotransmission in human isolated bladder smooth muscles. Exp Gerontol. 2001;36:99-109. 12. Birder LA, Ruan HZ, Chopra B, et al. Alterations in P2X and P2Y purinergic receptor expression in urinary bladder from normal cats and cats with interstitial cystitis. Am J Physiol Renal Physiol. 2004; 287:F1084-F1091.
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13. Kim JC, Yoo JS, Park EY, et al. Muscarinic and purinergic receptor expression in the urothelium of rats with detrusor overactivity induced by bladder outlet obstruction. BJU Int. 2008;101:371-375. 14. Wirkner K, K€ ofalvi A, Fischer W, et al. Supersensitivity of P2X receptors in cerebrocortical cell cultures after in vitro ischemia. J Neurochem. 2005;95:1421-1437. 15. Milano PM, Douillet CD, Riesenman PJ, et al. Intestinal ischemiareperfusion injury alters purinergic receptor expression in clinically relevant extraintestinal organs. J Surg Res. 2008;145:272-278. 16. Azadzoi KM, Chen B, Radisavljevic Z, Mike Siroky. Molecular reactions and ultrastructural damage in the chronically ischemic bladder. J Urol. 2011;186:2115-2122. 17. Kennedy C, Tasker PN, Gallacher G, et al. Identification of atropine- and P2X1 receptor antagonist-resistant, neurogenic contractions of the urinary bladder. J Neurosci. 2007;27:845-851. 18. Elneil S, Skepper JN, Kidd EJ, et al. Distribution of P2X(1) and P2X(3) receptors in the rat and human urinary bladder. Pharmacology. 2001;63:120-128. 19. Cockayne DA, Hamilton SG, Zhu QM, et al. Urinary bladder hyporeflexia and reduced pain-related behaviour in P2X3-deficient mice. Nature. 2000;407:1011-1015. 20. Lee HY, Bardini M, Burnstock G. Distribution of P2X receptors in the urinary bladder and the ureter of the rat. J Urol. 2000;163:20022007. 21. Ferguson DR, Kennedy I, Burton TJ. ATP is released from rabbit urinary bladder epithelial cells by hydrostatic pressure changesea possible sensory mechanism? J Physiol. 1997;505:503-511.
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22. Cockayne DA, Dunn PM, Zhong Y, et al. P2X2 knockout mice and P2X2/P2X3 double knockout mice reveal a role for the P2X2 receptor subunit in mediating multiple sensory effects of ATP. J Physiol. 2005;567:621-639. 23. Azadzoi KM, Yalla SV, Siroky MB. Human bladder smooth muscle damage in disturbed oxygen tension. Urology. 2011;78:967.e9-967. e15. 24. Kershen RT, Azadzoi KA, Siroky MB. Blood flow, pressure and compliance in the male human bladder. J Urol. 2002;168:121-125. 25. Judkins CP, Sobey CG, Dang TT, et al. NADPH-induced contractions of mouse aorta do not involve NADPH oxidase: a role for P2X receptors. J Pharmacol Exp Ther. 2006;317:644-650. 26. O’Reilly BA, Kosaka AH, Knight GF, et al. P2X receptors and their role in female idiopathic detrusor instability. J Urol. 2002;167: 157-164. 27. Yoshida M, Miyamae K, Iwashita H, et al. Management of detrusor dysfunction in the elderly: changes in acetylcholine and adenosine triphosphate release during aging. Urology. 2004;63:17-23. 28. Moore KH, Ray FR, Barden JA. Loss of purinergic P2X(3) and P2X(5) receptors innervation in human detrusor from adults with urge incontinence. J Neurosci. 2001;21:RCS166. 29. Brady CM, Apostolidis A, Yiangou Y, et al. P2X3-immunoreactive nerve fibres in neurogenic detrusor overactivity and the effect of intravesical resiniferatoxin. Eur Urol. 2004;46:247-253. 30. Ray FR, Moore KH, Hansen MA, et al. Loss of purinergic P2X receptor innervation in human detrusor and subepithelium from adults with sensory urgency. Cell Tissue Res. 2003;314:351-359.
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