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The Role of the Urothelium and ATP in Mediating Detrusor Smooth Muscle Contractility
Basic and Translational Science The Role of the Urothelium and ATP in Mediating Detrusor Smooth Muscle Contractility Aneira Gracia Hidayat Santoso, Ika Ariyani Bte Sonarno, Noor Aishah Bte Arsad, and Willmann Liang OBJECTIVES
METHODS
RESULTS
CONCLUSIONS
To examine the contractility of urothelium-intact (⫹UE) and urothelium-denuded (–UE) rat detrusor strips under adenosine triphosphate (ATP) treatment. Purinergic signaling exists in the bladder but both the inhibitory effect of ATP on detrusor contractions and the function of urothelial ATP are not established. Detrusor strips were obtained from bladders of young adult rats. Isometric tension from both transverse and longitudinal contractions was measured using a myograph. The muscarinic agonist carbachol (CCh) was used to induce contractions, which were under the influences of different concentrations of ATP. In both ⫹UE and –UE strips, 1 mM ATP suppressed CCh-induced contractions. In longitudinal contractions, ATP added to the inhibitory effect of urothelium on CCh responses. Removal of the urothelium, but with exogenous ATP added, recovered the CCh responses to the same level as in ⫹UE strips with no added ATP. Transverse contractions were less susceptible to ATP in the presence of urothelium. We showed that the urothelium and ATP suppressed CCh-induced contractions to a similar extent. The findings suggest an inhibitory role of urothelial ATP in mediating detrusor smooth muscle contractility, which may be impaired in diseased bladders. UROLOGY 76: 1267.e7–1267.e12, 2010. © 2010 Elsevier Inc.
E
fferent control of micturition is under the innervation of parasympathetic nerves on the bladder.1 Both acetylcholine and adenosine triphosphate (ATP) are coreleased as neurotransmitters.2 The urothelium releases more ATP in turn when P2X and P2Y receptors are activated.3 Together, neural and urothelial ATP stimulate P2X and P2Y receptors on detrusor smooth muscle to induce, respectively, contractions and relaxations.4-7 Although purinergic control is less important than that of cholinergic in normal conditions, ATP neurotransmission becomes more significant in bladder diseases.8 The healthy urothelium releases yet unidentifiable substances that diminish detrusor smooth muscle contractility, preventing their overactivity and consequent involuntary urine loss.9,10 Conversely, a dysfunctional or damaged urothelial layer is often implicated in bladder diseases. It has been reported that increased urothelial permeability or inhibition of ecto-ATPase activity, both observed in overactive bladder, renders the detrusor smooth muscle more responsive to ATP.11 The From the School of Biological Sciences, Nanyang Technological University, Singapore, Singapore Reprint requests: Willmann Liang, School of Biological Sciences, Nanyang Technological University, Singapore 637551, Singapore. E-mail: [email protected] Submitted: April 3, 2010, accepted (with revisions): June 22, 2010
© 2010 Elsevier Inc. All Rights Reserved
result is an increase in unwanted bladder spasms as well as bladder pain, the latter a result of increased sensory input to the central nervous system.3 Whether ATP also functions as an inhibitory factor in detrusor smooth muscle, particularly in normal, healthy bladder, remains questionable. To gain more insight into urothelium-mediated inhibitory control of detrusor contractions, the present study examines the effects of ATP on carbachol (CCh)induced contractions in urothelium-intact (⫹UE) and urothelium-denuded (–UE) rat detrusor strips. Considering reports of directional differences in contractile sensitivity to pharmacologic stimuli, both transverse and longitudinal contractions are measured in this study.
MATERIAL AND METHODS Tissue Preparation All procedures were performed according to rules outlined by the Institutional Animal Care and Use Committee at Nanyang Technological University, Singapore (Project approval No: ARF SBS/NIE-A 003). Six to seven-week-old Sprague-Dawley rats of either gender were killed by CO2 asphyxiation. The whole bladder was harvested as previously described and immediately placed in carbogen-aerated ice-cold Krebs’ solution.12 The bladder base, which made up about one third of the bladder, was discarded. Only tissues isolated from the bladder 0090-4295/10/$36.00 1267.e7 doi:10.1016/j.urology.2010.06.040
dome (detrusor) were used. The bladder dome was cut open along the lateral sides and the urothelium was exposed. Fine pins were used to fix the tissue on a Sylgard-coated Petri dish. Using a razor blade, 4 strips measuring 5 ⫻ 1 mm each were dissected from the detrusor.13 Two of the strips were cut with the longer side parallel to the longitudinal axis of the detrusor. Two of the strips were cut with the longer side parallel to the transverse axis of the detrusor. The longer side of the strip was in line with the direction of the contractile force measurement. Both urothelium-intact (⫹UE) and urothelium-denuded (–UE) strips were used. In –UE strips, the urothelium was carefully excised with fine dissecting scissors. All strips were mounted on a tissue myograph system (Danish Myo Technology Model 800 MS, Aarhus, Denmark) containing Krebs’ solution at 37°C. Isometric tension was monitored in both transverse and longitudinal directions and recorded using a Powerlab interface and LabChart software (ADInstruments, Bella Vista, Australia).
Experimental Protocol The detrusor strips were allowed to equilibrate for 30 minutes with multiple washouts at 2 g of resting tension. During the equilibration and subsequent washout periods, the resting tension was consistently adjusted to 2 g. The viability of the strips was tested using K⫹-Krebs’ solution bubbled with a mixture of 95% oxygen and 5% carbon dioxide. After an additional 30 minutes of continuous washout, cumulative concentration-response curves of the muscarinic agonist CCh were constructed with and without ATP pretreatment for 5 minutes. Application of ATP (0.01-1 mM) yielded a rapid, transient contractile response. The resting tension was again adjusted to 2 g before subsequent additions of CCh. Thirty minutes of continuous washout with Krebs’ solution were allowed in between each set of ATP-CCh stimulation. Internal time-matched controls of CCh responses were performed by randomizing the order of the pretreatment conditions of 0 (control), 0.01, 0.1, and 1 mM ATP over the course of the day. Figure 1 shows typical tracings of control and 1 mM ATP-pretreated CCh-induced contractions. Strips from 7 animals were used in transverse contractions; 8 in longitudinal contractions.
Drugs and Chemicals The composition of Krebs’ solution was as follows (in mM): NaCl (119), MgCl2 (1.2), NaH2PO4 (1.2), NaHCO3 (15), KCl (4.6), CaCl2 (1.5), and glucose (11). For K⫹-Krebs’ solution, no NaCl was added but 124 mM KCl was used instead. All constituents remained the same otherwise. All chemicals and drugs used in this study were purchased from Sigma-Aldrich, Co. (Singapore). All drugs were dissolved in Ca2⫹-free Krebs’ solution.
Statistical Analysis Calculations and statistical analysis were done using the prism 4 software (GraphPad Software, Inc., La Jolla, CA). The peak tension elicited by each addition of CCh in individual detrusor strips was used in subsequent calculations. Raw values were normalized to dry tissue weights, measured after the experiments, so that weight-insensitive contractile force data could be obtained. Maximal CCh-induced contraction in the –UE strip was considered as 100% contraction. In consideration of the phasic contractions sometimes observed, an average value was obtained over a 10-second interval (from LabChart) after each CCh addition had reached a steady-state. This usually applied 1267.e8
Figure 1. Sample tracings of carbachol (CCh)-induced contractions with and without 1 mM ATP pretreatment. Isometric tension was measured in (A) transverse and (B) longitudinal directions in both urothelium-intact (⫹UE, black dashed line) and urothelium-denuded (–UE, gray solid line) detrusor strips. Five concentrations of CCh were added in a cumulative manner as follows (in M): (i) 0.01, (ii) 0.1, (iii) 1, (iv) 10, (v) 100. The peak tension elicited by each addition of CCh was analyzed to generate the average data presented in Figs. 2 and 3 and Table 1. to the lower CCh concentrations, where the resultant force development was rather slow, compared with the rapid contraction induced by increasing amounts of CCh. The average values were used for statistical analyses as follows. Concentrationdependent effects of ATP on CCh (10 M) responses were analyzed with one-way analysis of variance (ANOVA) and Bonferroni post hoc test. Two-way ANOVA and Bonferroni post hoc test were used to compare amplitudes of CCh-induced contractions with and without ATP and/or urothelium. All data shown in graphs and tables were mean values ⫾ SEM. P values of less than .05 (P ⬍.05) were considered statistically different.
RESULTS The Greater Contractility to CCh of –UE Compared With ⴙUE Detrusor Strips was Preserved in the Presence of ATP Detrusor strips with ⫹UE and –UE were treated with 3 concentrations of ATP (0.01, 0.1, and 1 mM), each followed by cumulative additions of CCh to induce contractions. Table 1 compares the responses to 10 M UROLOGY 76 (5), 2010
Table 1. Effects of ATP pretreatment on carbachol (CCh)-induced transverse and longitudinal contractions in rat detrusor strips Transverse (n ⫽ 7)
Longitudinal (n ⫽ 8)
(ATP) (mM)
⫹UE
–UE
⫹UE
–UE
0 (Control) 0.01 0.1 1
60.45 ⫾ 7.46 67.28 ⫾ 9.43 68.75 ⫾ 5.05 49.94 ⫾ 10.00
86.24 ⫾ 2.71* 91.12 ⫾ 4.96* 87.77 ⫾ 1.89* 62.69 ⫾ 6.66†
64.71 ⫾ 5.73 70.26 ⫾ 3.78 68.23 ⫾ 4.18 43.22 ⫾ 6.73†
86.79 ⫾ 2.39* 84.41 ⫾ 4.45* 86.94 ⫾ 1.87* 59.80 ⫾ 8.14*†
Both urothelium-intact (⫹UE) and urothelium-denuded (–UE) strips are included. Responses to 10 M CCh, equating roughly 60% to 80% of contractility, are compared among the various ATP pretreatments, ranging from 0.01–1 mM. The responses are expressed as percentages of maximal contraction in the denuded tissue. n indicates the number of animals used. * P ⬍.05 vs ⫹UE tissues under the same ATP pretreatment. † P ⬍.05 vs control.
Figure 2. Inhibitory effects of urothelium and ATP pretreatment on CCh-induced contractions in (A) transverse (n ⫽ 7) and (B) longitudinal (n ⫽ 8) directions. Contractility of urothelium-intact (⫹UE) strips were measured both in the presence (open squares) and absence (closed squares) of 1 mM ATP. –UE strips were not pretreated with ATP (closed circles). Both transverse and longitudinal contractions to CCh stimulation were greater in –UE than ⫹UE strips with or without ATP (*P ⬍.05 vs ⫹UE strips). Only longitudinal contractions showed further suppression of CCh responses in ⫹UE strips when ATP was present (⫹P ⬍.05 vs ⫹UE strips). Exogenous ATP enhanced the inhibitory effect of urothelium in CCh-induced longitudinal contractions.
CCh, which induced contractions to 60% to 80% maximum in general, under various ATP pretreatments. It is evident that CCh-induced contractions were smaller in ⫹UE than –UE strips, regardless of the presence of ATP. The only exception is the transverse contractions, where after 1-mM ATP treatment, the CCh responses were not significantly different between ⫹UE and –UE strips. Overall, it could be concluded that ATP (from 0.01-1 mM) exerted a similar inhibitory effect as the urothelium in decreasing contractility to CCh. CCh-Induced Contractions Were Suppressed by ATP Pretreatment In Table 1, the ATP-pretreated CCh responses are compared with control values within the same column, ie, comparison among 0-1 mM ATP pretreatment within ⫹UE and –UE tissues, respectively. Except for 1 mM, all other concentrations of ATP failed to elicit any significant effects in CCh-induced contractions. In all detrusor strips (including both ⫹UE and –UE strips), 1 mM ATP UROLOGY 76 (5), 2010
was able to suppress the subsequent CCh-induced contractions in the longitudinal direction. In transverse contractions, CCh responses were significantly reduced by 1 mM ATP only when the urothelium was removed. Nonetheless, the effects of a high concentration of ATP (ie, 1 mM) were examined further. Inhibitory Effect of the Urothelium Was Enhanced by ATP in Longitudinal Contractions Only Figures 1 and 2 show the CCh concentration-response curves of both transverse and longitudinal contractions in ⫹UE strips (with and without ATP) and –UE strips. In both transverse and longitudinal contractions, CCh responses in the absence of ATP were smaller in ⫹UE than in –UE strips, indicating inhibitory effects of the urothelium. Transverse contractions were unaltered by ATP pretreatment in ⫹UE strips (Fig. 2A). By contrast, longitudinal contractions were suppressed in ⫹UE strips under the same condition, as shown by the additional suppression of the CCh concentration-response curve 1267.e9
Figure 3. Similar inhibition exerted by urothelium and ATP on CCh-induced contractions in (A) transverse (n ⫽ 7) and (B) longitudinal (n ⫽ 8) directions. Contractility of ⫹UE strips were measured both in the presence (open squares) and absence (closed squares) of 1 mM ATP. –UE strips were pretreated with ATP (open circles). In both transverse and longitudinal contractions, exogenous ATP suppressed CCh responses in –UE strips to the same level as in ⫹UE strips (without ATP) as shown by the overlapping traces. Differences were detected in ATP-pretreated CCh-induced longitudinal contractions between ⫹UE and –UE strips (⫹P ⬍.05 vs ⫹UE strips with ATP). Exogenous ATP in –UE strips restored the inhibition exerted by urothelium (in ⫹UE strips) but fell short of suppressing CCh responses further, as seen in ⫹UE strips with ATP (*P ⬍.05 vs ⫹UE strips).
(Fig. 2B). Thus, ATP pretreatment enhanced the inhibitory effects on CCh responses in longitudinal contractions only. Contractions Under ATP Treatment in –UE Strips Were Restored to the Same Level as in ⴙUE Strips It was shown in Figure 2B that ATP added to the inhibitory effect of the urothelium in longitudinal contractions only. In contrast, the effect of ATP in –UE strips was the same in both transverse and longitudinal contractions. Figure 3 shows the CCh concentration-response curves of contractions in ⫹UE strips (with and without ATP) and –UE strips (with ATP). Both transverse and longitudinal contractions to CCh stimulation were recovered in –UE strips pretreated with ATP to the same level as if the urothelium was intact (ie, in ⫹UE strips without ATP) (Fig. 3). Exogenous ATP added to –UE strips, however, was not able to suppress CCh responses to the same extent as in ⫹UE strips with ATP, as shown in the differences in longitudinal contractions.
COMMENT The contractile effect of ATP on detrusor smooth muscle has been reported by many.4-6 In response to bladder distension, the urothelium releases ATP to induce bladder contractions,3 an observation also made in our study (data not shown). However, there is very limited literature documenting the relaxant effect of ATP, exemplified by the sole report of McMurray et al. in which both contractile and relaxant effects of ATP in urotheliumdenuded (–UE) detrusor strips were observed.7 Expression of purinergic P2X and P2Y receptors on detrusor smooth muscle are postulated to participate in, respec1267.e10
tively, contractions and relaxations.2,14,15 Both P2X and P2Y receptors are also found on the urothelium,3,15 which respond to neural ATP release by releasing more ATP. In addition to stimulating contractions, ATP from the urothelial source may also inhibit contractions. Evidence was presented here for the first time, demonstrating the inhibitory effect of ATP on CCh-induced contractions in adult rat detrusor strips with and without urothelium. In addition, differential sensitivity to ATP was demonstrated between contractions in the transverse and longitudinal directions. Numerous studies have demonstrated that removal of the urothelium enhances the contractile responses to CCh in the detrusor of different species, including the rat.9,16,17 Our results in Table 1, Figure 1 (dotted lines), and Figure 2 (solid lines) also support the inhibitory role of the urothelium in CCh-induced contractions. Two additional observations are made from the values in Table 1. First, pretreatment with ATP (from 0.01-1 mM) preserved the greater contractility seen in the urotheliumdenuded (–UE) strips compared with the urotheliumintact (⫹UE) ones. Second, a high concentration of ATP (at 1 mM) also significantly suppressed the subsequent CCh-induced contractions in both ⫹UE and –UE strips. Thus, ATP exerts an inhibitory effect on CChinduced contractions by acting directly on detrusor smooth muscle (in –UE strips). At the same time, exogenous ATP may also stimulate the urothelium to release more ATP,3 which in turn inhibits detrusor contractions (in ⫹UE strips). The two putative roles of ATP (at 1 mM) on the urothelium and detrusor smooth muscle converge to support the inhibitory effect on CCh-induced contractions. UROLOGY 76 (5), 2010
From Figure 2B, the inhibitory effect of ATP on longitudinal CCh responses can be deduced from the largely overlapping traces representing ⫹UE and –UE (with ATP) strips. These two traces indicate that the presence of ATP in –UE strips render them to respond to CCh stimulation as if the urothelium were intact (ie, as ⫹UE strips). The amount of ATP added to the –UE strips was thus enough to make up for the loss of urothelium in mediating longitudinal contractions. When ATP was added to ⫹UE strips (Fig. 2B), a further suppression of CCh-induced longitudinal contractions resulted. The findings in Figures 2B and 3B collectively could be explained by the following postulation. The detrusor smooth muscle may receive inhibitory input directly from exogenous ATP and indirectly from endogenous ATP released by the urothelium. Similar to longitudinal contractions, exogenous ATP acts directly on detrusor smooth muscle in –UE strips, suppressing transverse contractions to the same extent as if the urothelium were present (Fig. 3A). However, ATP pretreatment did not suppress CCh responses any further in ⫹UE strips (Fig. 2A). Thus, unlike in longitudinal contractions, exogenous ATP did not provide additional inhibition on top of the urothelium in transverse contractions. Based on these findings, it can be concluded that transverse contractions are less sensitive to the inhibitory effect of ATP, especially in ⫹UE strips. That ATP suppressed CCh-induced longitudinal but not transverse contractions in ⫹UE strips reiterates the importance in measuring contractility in more than one direction. Depending on the stimuli, responses could vary between contractions measured in different directions.13,18-21 For example, it has been reported that transverse contractions are more sensitive than longitudinal contractions to certain K⫹ channel blockers but not others.21 On the contrary, longitudinal contractions are more responsive to exogenous ATP, suggesting a greater susceptibility of detrusor smooth muscle contracting in this direction. The control exerted by the urothelium in ⫹UE strips, however, is indistinguishable between transverse and longitudinal contractions. To account for any potential variations, it is therefore of interest to always compare directional contractile differences in organs like the bladder, where contractions in multiple directions occur. We demonstrated that ATP pretreatment leads to diminished contractility to CCh stimulation. In longitudinal contractions, the presence of ATP added to the inhibitory effect in both ⫹UE and –UE strips. Particularly in –UE strips, exogenous ATP restored the contractile level to that in ⫹UE strips. The restored CCh responses were also observed in transverse contractions. It may be speculated that the exogenous ATP replaces the endogenous secretion that would have come from the urothelium. The use of suramin, a nonselective P2X and P2Y receptor blocker, has proven ineffective in ⫹UE strips in identifying, if any, the urothelial ATP release UROLOGY 76 (5), 2010
component.9 The negative finding of suramin may be attributed to the expression of both P2X and P2Y receptor families in both the urothelium and detrusor smooth muscle. Even if a selective P2Y receptor blocker becomes available, complications will arise from the inability to block the receptors in either the urothelium or detrusor smooth muscle specifically. Therefore, the existing data may be the strongest to date, indicating the inhibitory function of urothelial ATP on detrusor smooth muscle. The functional relationship between urothelial P2X and P2Y receptors may also correlate with detrusor smooth muscle contractility.3 Blocking one or another receptor family in the urothelium may affect detrusor contractions to a different extent. Future investigations may focus on determining the relative importance of the various urothelial purinergic receptor subtypes in mediating ATP release, and consequently bladder contractility. Because a stronger component of urothelial ATP release is also observed in bladder pathologies,8,22-24 further studies may examine disease-related changes associated with ATP effects on the urothelium or detrusor smooth muscle. Our data on normal rat detrusor strips may provide valuable groundwork for comparison with the diseased bladders also of rat origin, eg, overactive bladder model in spontaneously hypertensive rats.25,26 Findings from animal models of bladder disorders may subsequently be applied to more clinically relevant studies using human bladder samples. In addition to examining human detrusor contractility directly, the contractile mediating effect of human urothelial ATP on the rat detrusor smooth muscle can also be assessed by using a superfusate bioassay system.
CONCLUSIONS We showed in normal rat detrusor strips that ATP may originate from the urothelium and inhibit contractions. A greater influence of ATP on longitudinal contractions was observed. In particular, exogenous ATP enhanced the urothelium-mediated inhibition of longitudinal contractions. Altogether a good balance between the contractile and relaxant effect of ATP may implicate normal bladder physiology. Based on the present findings, it will be of considerable interest to investigate disease-mediated alterations in urothelial ATP release and detrusor contractility in the future, using bladders from animal diseased models and from humans. Acknowledgments. This study was supported by grants from the Singapore Ministry of Education (RG63/06 and RG83/07) and Nanyang Technological University (SUG15/07).
References 1. Andersson KE, Arner A. Urinary bladder contraction and relaxation: physiology and pathophysiology. Physiol Res. 2004;84:935986. 2. Fry CH, Meng E, Young JS. The physiological function of lower urinary tract smooth muscle. Auton Neurosci. 2010;154:3-13. 3. Birder LA. Urothelial signaling. Auton Neurosci. 2010;153:33-40.
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4. Burnstock G, Dumsday B, Smythe A. Atropine resistant excitation of the urinary bladder: the possibility of transmission via nerves releasing a purin nucleotide. Br J Pharmacol. 1972;44:451-461. 5. Hoyle CHV, Chapple C, Burnstock G. Isolated human bladder: evidence for an adenine dicucleotide acting on P2X-purinoceptors and for purinergic transmission. Eur J Pharmacol. 1989;174:115118. 6. Usune S, Katsuragi T, Furukawa T. Effects of PPADS and suramin on contractions and cytoplasmic Ca2⫹ changes evoked by AP. 4 A, ATP and ␣, -methylene ATP in guinea-pig urinary bladder. Br J Pharmacol. 1996;117:698-702. 7. McMurray G, Dass N, Brading A. Purinoceptor subtypes mediating contraction and relaxation of marmoset urinary bladder smooth muscle. Br J Pharmacol. 1998;123:1579-1586. 8. Ford APDW, Gever JR, Nunn PA, et al. Purinoceptors as therapeutic targets for lower urinary tract dysfunction. Br J Pharmacol. 2006;147:S132-S143. 9. Hawthorn MH, Chapple CR, Cock M, et al. Urothelium-derived inhibitory factor(s) influences on detrusor muscle contractility in vitro. Br J Pharmacol. 2000;129:416-419. 10. Murakami S, Chapple C, Akino H, et al. The role of the urothelium in mediating bladder responses to isoprenaline. BJU Int. 2007;99:669-673. 11. Nishiguchi J, Hayashi Y, Chancellor MB, et al. Detrusor overactivity induced by intravesical application of adenosine 5’-triphosphate under different delivery conditions in the rat. Urology. 2005; 66:1332-1337. 12. Liang W, Afshar K, Stothers L, et al. The influence of ovariectomy and estrogen replacement on voiding patterns and detrusor muscarinic receptor affinity in the rat. Life Sci. 2002;71:351-362. 13. Lo WN, Liang W. Blockade of voltage-sensitive K⫹ channels increases contractility more in transverse than in longitudinal rat detrusor strips. Urology. 2009;73:400-404. 14. Elneil S, Skepper JN, Kidd EJ, et al. Distribution of P2X1 and P2X3 receptors in the rat and human urinary bladder. Pharmacologist. 2001;63:120-128.
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15. Chopra B, Gever J, Barrick SR, et al. Expression and function of rat urothelial P2Y receptors. Am J Physiol Ren Physiol. 2008;294:F821F829. 16. Wuest M, Kaden S, Hakenberg OW, et al. Effects of Rilmakalim on detrusor contraction in the presence and absence of urothelium. Naunyn-Schmiedeberg Arch. Pharmacologist. 2005;372:203-212. 17. Chaiyaprasithi B, Mang CF, Kilbinger H, et al. Inhibition of human detrusor contraction by a urothelium derived factor. J Urol. 2003;170:1897-1900. 18. Potjer RM, Constantinou CE. Frequency of spontaneous contractions in longitudinal and transverse bladder strips. Am J Physiol. 1989;257:R781-R787. 19. Pagala MK, Tetsoti L, Nagpal D, et al. Aging effects on contractility of longitudinal and circular detrusor and trigone of rat bladder. J Urol. 2001;166:721-727. 20. Pagala M, Lehman DS, Morgan MP, et al. Physiological fatigue of smooth muscle contractions in rat urinary bladder. BJU Int. 2006; 97:1087-1093. 21. Lo WN, Santoso AGH, Liang W. Differences in transverse and longitudinal rat detrusor contractility under K⫹ channel blockade. UroToday Int J. 2010;3. doi:10.3834/uij.1944-5784.2010.04.07. 22. Birder LA, Barrick SR, Roppolo JR, et al. Feline interstitial cystitis results in mechanical hypersensitivity and altered ATP release from bladder urothelium. Am J Physiol Ren Physiol. 2003;285:F423-F429. 23. Sun Y, Keay S, DeDeyne P, et al. Activated adenosine triphosphate release from bladder uroepithelial cells in patients with interstitial cystitis. J Urol. 2001;166:1951-1956. 24. Harvey R. The contractile potency of adenosine triphosphate and ecto-adenosine triphosphatase activity in guinea pig detrusor and detrusor from patients with a stable, unstable or obstructed bladder. J Urol. 2002;168:1235-1239. 25. McMurray G, Casey JH, Naylor AM. Animal models in urological disease and sexual dysfunction. Br J Pharmacol. 2006;147:S62-S79. 26. Michel MC, Barendrecht MM. Physiological and pathological regulation of the autonomic control of urinary bladder contractility. Pharmacol Ther. 2008;117:297-312.
UROLOGY 76 (5), 2010
METHODS
RESULTS
CONCLUSIONS
To examine the contractility of urothelium-intact (⫹UE) and urothelium-denuded (–UE) rat detrusor strips under adenosine triphosphate (ATP) treatment. Purinergic signaling exists in the bladder but both the inhibitory effect of ATP on detrusor contractions and the function of urothelial ATP are not established. Detrusor strips were obtained from bladders of young adult rats. Isometric tension from both transverse and longitudinal contractions was measured using a myograph. The muscarinic agonist carbachol (CCh) was used to induce contractions, which were under the influences of different concentrations of ATP. In both ⫹UE and –UE strips, 1 mM ATP suppressed CCh-induced contractions. In longitudinal contractions, ATP added to the inhibitory effect of urothelium on CCh responses. Removal of the urothelium, but with exogenous ATP added, recovered the CCh responses to the same level as in ⫹UE strips with no added ATP. Transverse contractions were less susceptible to ATP in the presence of urothelium. We showed that the urothelium and ATP suppressed CCh-induced contractions to a similar extent. The findings suggest an inhibitory role of urothelial ATP in mediating detrusor smooth muscle contractility, which may be impaired in diseased bladders. UROLOGY 76: 1267.e7–1267.e12, 2010. © 2010 Elsevier Inc.
E
fferent control of micturition is under the innervation of parasympathetic nerves on the bladder.1 Both acetylcholine and adenosine triphosphate (ATP) are coreleased as neurotransmitters.2 The urothelium releases more ATP in turn when P2X and P2Y receptors are activated.3 Together, neural and urothelial ATP stimulate P2X and P2Y receptors on detrusor smooth muscle to induce, respectively, contractions and relaxations.4-7 Although purinergic control is less important than that of cholinergic in normal conditions, ATP neurotransmission becomes more significant in bladder diseases.8 The healthy urothelium releases yet unidentifiable substances that diminish detrusor smooth muscle contractility, preventing their overactivity and consequent involuntary urine loss.9,10 Conversely, a dysfunctional or damaged urothelial layer is often implicated in bladder diseases. It has been reported that increased urothelial permeability or inhibition of ecto-ATPase activity, both observed in overactive bladder, renders the detrusor smooth muscle more responsive to ATP.11 The From the School of Biological Sciences, Nanyang Technological University, Singapore, Singapore Reprint requests: Willmann Liang, School of Biological Sciences, Nanyang Technological University, Singapore 637551, Singapore. E-mail: [email protected] Submitted: April 3, 2010, accepted (with revisions): June 22, 2010
© 2010 Elsevier Inc. All Rights Reserved
result is an increase in unwanted bladder spasms as well as bladder pain, the latter a result of increased sensory input to the central nervous system.3 Whether ATP also functions as an inhibitory factor in detrusor smooth muscle, particularly in normal, healthy bladder, remains questionable. To gain more insight into urothelium-mediated inhibitory control of detrusor contractions, the present study examines the effects of ATP on carbachol (CCh)induced contractions in urothelium-intact (⫹UE) and urothelium-denuded (–UE) rat detrusor strips. Considering reports of directional differences in contractile sensitivity to pharmacologic stimuli, both transverse and longitudinal contractions are measured in this study.
MATERIAL AND METHODS Tissue Preparation All procedures were performed according to rules outlined by the Institutional Animal Care and Use Committee at Nanyang Technological University, Singapore (Project approval No: ARF SBS/NIE-A 003). Six to seven-week-old Sprague-Dawley rats of either gender were killed by CO2 asphyxiation. The whole bladder was harvested as previously described and immediately placed in carbogen-aerated ice-cold Krebs’ solution.12 The bladder base, which made up about one third of the bladder, was discarded. Only tissues isolated from the bladder 0090-4295/10/$36.00 1267.e7 doi:10.1016/j.urology.2010.06.040
dome (detrusor) were used. The bladder dome was cut open along the lateral sides and the urothelium was exposed. Fine pins were used to fix the tissue on a Sylgard-coated Petri dish. Using a razor blade, 4 strips measuring 5 ⫻ 1 mm each were dissected from the detrusor.13 Two of the strips were cut with the longer side parallel to the longitudinal axis of the detrusor. Two of the strips were cut with the longer side parallel to the transverse axis of the detrusor. The longer side of the strip was in line with the direction of the contractile force measurement. Both urothelium-intact (⫹UE) and urothelium-denuded (–UE) strips were used. In –UE strips, the urothelium was carefully excised with fine dissecting scissors. All strips were mounted on a tissue myograph system (Danish Myo Technology Model 800 MS, Aarhus, Denmark) containing Krebs’ solution at 37°C. Isometric tension was monitored in both transverse and longitudinal directions and recorded using a Powerlab interface and LabChart software (ADInstruments, Bella Vista, Australia).
Experimental Protocol The detrusor strips were allowed to equilibrate for 30 minutes with multiple washouts at 2 g of resting tension. During the equilibration and subsequent washout periods, the resting tension was consistently adjusted to 2 g. The viability of the strips was tested using K⫹-Krebs’ solution bubbled with a mixture of 95% oxygen and 5% carbon dioxide. After an additional 30 minutes of continuous washout, cumulative concentration-response curves of the muscarinic agonist CCh were constructed with and without ATP pretreatment for 5 minutes. Application of ATP (0.01-1 mM) yielded a rapid, transient contractile response. The resting tension was again adjusted to 2 g before subsequent additions of CCh. Thirty minutes of continuous washout with Krebs’ solution were allowed in between each set of ATP-CCh stimulation. Internal time-matched controls of CCh responses were performed by randomizing the order of the pretreatment conditions of 0 (control), 0.01, 0.1, and 1 mM ATP over the course of the day. Figure 1 shows typical tracings of control and 1 mM ATP-pretreated CCh-induced contractions. Strips from 7 animals were used in transverse contractions; 8 in longitudinal contractions.
Drugs and Chemicals The composition of Krebs’ solution was as follows (in mM): NaCl (119), MgCl2 (1.2), NaH2PO4 (1.2), NaHCO3 (15), KCl (4.6), CaCl2 (1.5), and glucose (11). For K⫹-Krebs’ solution, no NaCl was added but 124 mM KCl was used instead. All constituents remained the same otherwise. All chemicals and drugs used in this study were purchased from Sigma-Aldrich, Co. (Singapore). All drugs were dissolved in Ca2⫹-free Krebs’ solution.
Statistical Analysis Calculations and statistical analysis were done using the prism 4 software (GraphPad Software, Inc., La Jolla, CA). The peak tension elicited by each addition of CCh in individual detrusor strips was used in subsequent calculations. Raw values were normalized to dry tissue weights, measured after the experiments, so that weight-insensitive contractile force data could be obtained. Maximal CCh-induced contraction in the –UE strip was considered as 100% contraction. In consideration of the phasic contractions sometimes observed, an average value was obtained over a 10-second interval (from LabChart) after each CCh addition had reached a steady-state. This usually applied 1267.e8
Figure 1. Sample tracings of carbachol (CCh)-induced contractions with and without 1 mM ATP pretreatment. Isometric tension was measured in (A) transverse and (B) longitudinal directions in both urothelium-intact (⫹UE, black dashed line) and urothelium-denuded (–UE, gray solid line) detrusor strips. Five concentrations of CCh were added in a cumulative manner as follows (in M): (i) 0.01, (ii) 0.1, (iii) 1, (iv) 10, (v) 100. The peak tension elicited by each addition of CCh was analyzed to generate the average data presented in Figs. 2 and 3 and Table 1. to the lower CCh concentrations, where the resultant force development was rather slow, compared with the rapid contraction induced by increasing amounts of CCh. The average values were used for statistical analyses as follows. Concentrationdependent effects of ATP on CCh (10 M) responses were analyzed with one-way analysis of variance (ANOVA) and Bonferroni post hoc test. Two-way ANOVA and Bonferroni post hoc test were used to compare amplitudes of CCh-induced contractions with and without ATP and/or urothelium. All data shown in graphs and tables were mean values ⫾ SEM. P values of less than .05 (P ⬍.05) were considered statistically different.
RESULTS The Greater Contractility to CCh of –UE Compared With ⴙUE Detrusor Strips was Preserved in the Presence of ATP Detrusor strips with ⫹UE and –UE were treated with 3 concentrations of ATP (0.01, 0.1, and 1 mM), each followed by cumulative additions of CCh to induce contractions. Table 1 compares the responses to 10 M UROLOGY 76 (5), 2010
Table 1. Effects of ATP pretreatment on carbachol (CCh)-induced transverse and longitudinal contractions in rat detrusor strips Transverse (n ⫽ 7)
Longitudinal (n ⫽ 8)
(ATP) (mM)
⫹UE
–UE
⫹UE
–UE
0 (Control) 0.01 0.1 1
60.45 ⫾ 7.46 67.28 ⫾ 9.43 68.75 ⫾ 5.05 49.94 ⫾ 10.00
86.24 ⫾ 2.71* 91.12 ⫾ 4.96* 87.77 ⫾ 1.89* 62.69 ⫾ 6.66†
64.71 ⫾ 5.73 70.26 ⫾ 3.78 68.23 ⫾ 4.18 43.22 ⫾ 6.73†
86.79 ⫾ 2.39* 84.41 ⫾ 4.45* 86.94 ⫾ 1.87* 59.80 ⫾ 8.14*†
Both urothelium-intact (⫹UE) and urothelium-denuded (–UE) strips are included. Responses to 10 M CCh, equating roughly 60% to 80% of contractility, are compared among the various ATP pretreatments, ranging from 0.01–1 mM. The responses are expressed as percentages of maximal contraction in the denuded tissue. n indicates the number of animals used. * P ⬍.05 vs ⫹UE tissues under the same ATP pretreatment. † P ⬍.05 vs control.
Figure 2. Inhibitory effects of urothelium and ATP pretreatment on CCh-induced contractions in (A) transverse (n ⫽ 7) and (B) longitudinal (n ⫽ 8) directions. Contractility of urothelium-intact (⫹UE) strips were measured both in the presence (open squares) and absence (closed squares) of 1 mM ATP. –UE strips were not pretreated with ATP (closed circles). Both transverse and longitudinal contractions to CCh stimulation were greater in –UE than ⫹UE strips with or without ATP (*P ⬍.05 vs ⫹UE strips). Only longitudinal contractions showed further suppression of CCh responses in ⫹UE strips when ATP was present (⫹P ⬍.05 vs ⫹UE strips). Exogenous ATP enhanced the inhibitory effect of urothelium in CCh-induced longitudinal contractions.
CCh, which induced contractions to 60% to 80% maximum in general, under various ATP pretreatments. It is evident that CCh-induced contractions were smaller in ⫹UE than –UE strips, regardless of the presence of ATP. The only exception is the transverse contractions, where after 1-mM ATP treatment, the CCh responses were not significantly different between ⫹UE and –UE strips. Overall, it could be concluded that ATP (from 0.01-1 mM) exerted a similar inhibitory effect as the urothelium in decreasing contractility to CCh. CCh-Induced Contractions Were Suppressed by ATP Pretreatment In Table 1, the ATP-pretreated CCh responses are compared with control values within the same column, ie, comparison among 0-1 mM ATP pretreatment within ⫹UE and –UE tissues, respectively. Except for 1 mM, all other concentrations of ATP failed to elicit any significant effects in CCh-induced contractions. In all detrusor strips (including both ⫹UE and –UE strips), 1 mM ATP UROLOGY 76 (5), 2010
was able to suppress the subsequent CCh-induced contractions in the longitudinal direction. In transverse contractions, CCh responses were significantly reduced by 1 mM ATP only when the urothelium was removed. Nonetheless, the effects of a high concentration of ATP (ie, 1 mM) were examined further. Inhibitory Effect of the Urothelium Was Enhanced by ATP in Longitudinal Contractions Only Figures 1 and 2 show the CCh concentration-response curves of both transverse and longitudinal contractions in ⫹UE strips (with and without ATP) and –UE strips. In both transverse and longitudinal contractions, CCh responses in the absence of ATP were smaller in ⫹UE than in –UE strips, indicating inhibitory effects of the urothelium. Transverse contractions were unaltered by ATP pretreatment in ⫹UE strips (Fig. 2A). By contrast, longitudinal contractions were suppressed in ⫹UE strips under the same condition, as shown by the additional suppression of the CCh concentration-response curve 1267.e9
Figure 3. Similar inhibition exerted by urothelium and ATP on CCh-induced contractions in (A) transverse (n ⫽ 7) and (B) longitudinal (n ⫽ 8) directions. Contractility of ⫹UE strips were measured both in the presence (open squares) and absence (closed squares) of 1 mM ATP. –UE strips were pretreated with ATP (open circles). In both transverse and longitudinal contractions, exogenous ATP suppressed CCh responses in –UE strips to the same level as in ⫹UE strips (without ATP) as shown by the overlapping traces. Differences were detected in ATP-pretreated CCh-induced longitudinal contractions between ⫹UE and –UE strips (⫹P ⬍.05 vs ⫹UE strips with ATP). Exogenous ATP in –UE strips restored the inhibition exerted by urothelium (in ⫹UE strips) but fell short of suppressing CCh responses further, as seen in ⫹UE strips with ATP (*P ⬍.05 vs ⫹UE strips).
(Fig. 2B). Thus, ATP pretreatment enhanced the inhibitory effects on CCh responses in longitudinal contractions only. Contractions Under ATP Treatment in –UE Strips Were Restored to the Same Level as in ⴙUE Strips It was shown in Figure 2B that ATP added to the inhibitory effect of the urothelium in longitudinal contractions only. In contrast, the effect of ATP in –UE strips was the same in both transverse and longitudinal contractions. Figure 3 shows the CCh concentration-response curves of contractions in ⫹UE strips (with and without ATP) and –UE strips (with ATP). Both transverse and longitudinal contractions to CCh stimulation were recovered in –UE strips pretreated with ATP to the same level as if the urothelium was intact (ie, in ⫹UE strips without ATP) (Fig. 3). Exogenous ATP added to –UE strips, however, was not able to suppress CCh responses to the same extent as in ⫹UE strips with ATP, as shown in the differences in longitudinal contractions.
COMMENT The contractile effect of ATP on detrusor smooth muscle has been reported by many.4-6 In response to bladder distension, the urothelium releases ATP to induce bladder contractions,3 an observation also made in our study (data not shown). However, there is very limited literature documenting the relaxant effect of ATP, exemplified by the sole report of McMurray et al. in which both contractile and relaxant effects of ATP in urotheliumdenuded (–UE) detrusor strips were observed.7 Expression of purinergic P2X and P2Y receptors on detrusor smooth muscle are postulated to participate in, respec1267.e10
tively, contractions and relaxations.2,14,15 Both P2X and P2Y receptors are also found on the urothelium,3,15 which respond to neural ATP release by releasing more ATP. In addition to stimulating contractions, ATP from the urothelial source may also inhibit contractions. Evidence was presented here for the first time, demonstrating the inhibitory effect of ATP on CCh-induced contractions in adult rat detrusor strips with and without urothelium. In addition, differential sensitivity to ATP was demonstrated between contractions in the transverse and longitudinal directions. Numerous studies have demonstrated that removal of the urothelium enhances the contractile responses to CCh in the detrusor of different species, including the rat.9,16,17 Our results in Table 1, Figure 1 (dotted lines), and Figure 2 (solid lines) also support the inhibitory role of the urothelium in CCh-induced contractions. Two additional observations are made from the values in Table 1. First, pretreatment with ATP (from 0.01-1 mM) preserved the greater contractility seen in the urotheliumdenuded (–UE) strips compared with the urotheliumintact (⫹UE) ones. Second, a high concentration of ATP (at 1 mM) also significantly suppressed the subsequent CCh-induced contractions in both ⫹UE and –UE strips. Thus, ATP exerts an inhibitory effect on CChinduced contractions by acting directly on detrusor smooth muscle (in –UE strips). At the same time, exogenous ATP may also stimulate the urothelium to release more ATP,3 which in turn inhibits detrusor contractions (in ⫹UE strips). The two putative roles of ATP (at 1 mM) on the urothelium and detrusor smooth muscle converge to support the inhibitory effect on CCh-induced contractions. UROLOGY 76 (5), 2010
From Figure 2B, the inhibitory effect of ATP on longitudinal CCh responses can be deduced from the largely overlapping traces representing ⫹UE and –UE (with ATP) strips. These two traces indicate that the presence of ATP in –UE strips render them to respond to CCh stimulation as if the urothelium were intact (ie, as ⫹UE strips). The amount of ATP added to the –UE strips was thus enough to make up for the loss of urothelium in mediating longitudinal contractions. When ATP was added to ⫹UE strips (Fig. 2B), a further suppression of CCh-induced longitudinal contractions resulted. The findings in Figures 2B and 3B collectively could be explained by the following postulation. The detrusor smooth muscle may receive inhibitory input directly from exogenous ATP and indirectly from endogenous ATP released by the urothelium. Similar to longitudinal contractions, exogenous ATP acts directly on detrusor smooth muscle in –UE strips, suppressing transverse contractions to the same extent as if the urothelium were present (Fig. 3A). However, ATP pretreatment did not suppress CCh responses any further in ⫹UE strips (Fig. 2A). Thus, unlike in longitudinal contractions, exogenous ATP did not provide additional inhibition on top of the urothelium in transverse contractions. Based on these findings, it can be concluded that transverse contractions are less sensitive to the inhibitory effect of ATP, especially in ⫹UE strips. That ATP suppressed CCh-induced longitudinal but not transverse contractions in ⫹UE strips reiterates the importance in measuring contractility in more than one direction. Depending on the stimuli, responses could vary between contractions measured in different directions.13,18-21 For example, it has been reported that transverse contractions are more sensitive than longitudinal contractions to certain K⫹ channel blockers but not others.21 On the contrary, longitudinal contractions are more responsive to exogenous ATP, suggesting a greater susceptibility of detrusor smooth muscle contracting in this direction. The control exerted by the urothelium in ⫹UE strips, however, is indistinguishable between transverse and longitudinal contractions. To account for any potential variations, it is therefore of interest to always compare directional contractile differences in organs like the bladder, where contractions in multiple directions occur. We demonstrated that ATP pretreatment leads to diminished contractility to CCh stimulation. In longitudinal contractions, the presence of ATP added to the inhibitory effect in both ⫹UE and –UE strips. Particularly in –UE strips, exogenous ATP restored the contractile level to that in ⫹UE strips. The restored CCh responses were also observed in transverse contractions. It may be speculated that the exogenous ATP replaces the endogenous secretion that would have come from the urothelium. The use of suramin, a nonselective P2X and P2Y receptor blocker, has proven ineffective in ⫹UE strips in identifying, if any, the urothelial ATP release UROLOGY 76 (5), 2010
component.9 The negative finding of suramin may be attributed to the expression of both P2X and P2Y receptor families in both the urothelium and detrusor smooth muscle. Even if a selective P2Y receptor blocker becomes available, complications will arise from the inability to block the receptors in either the urothelium or detrusor smooth muscle specifically. Therefore, the existing data may be the strongest to date, indicating the inhibitory function of urothelial ATP on detrusor smooth muscle. The functional relationship between urothelial P2X and P2Y receptors may also correlate with detrusor smooth muscle contractility.3 Blocking one or another receptor family in the urothelium may affect detrusor contractions to a different extent. Future investigations may focus on determining the relative importance of the various urothelial purinergic receptor subtypes in mediating ATP release, and consequently bladder contractility. Because a stronger component of urothelial ATP release is also observed in bladder pathologies,8,22-24 further studies may examine disease-related changes associated with ATP effects on the urothelium or detrusor smooth muscle. Our data on normal rat detrusor strips may provide valuable groundwork for comparison with the diseased bladders also of rat origin, eg, overactive bladder model in spontaneously hypertensive rats.25,26 Findings from animal models of bladder disorders may subsequently be applied to more clinically relevant studies using human bladder samples. In addition to examining human detrusor contractility directly, the contractile mediating effect of human urothelial ATP on the rat detrusor smooth muscle can also be assessed by using a superfusate bioassay system.
CONCLUSIONS We showed in normal rat detrusor strips that ATP may originate from the urothelium and inhibit contractions. A greater influence of ATP on longitudinal contractions was observed. In particular, exogenous ATP enhanced the urothelium-mediated inhibition of longitudinal contractions. Altogether a good balance between the contractile and relaxant effect of ATP may implicate normal bladder physiology. Based on the present findings, it will be of considerable interest to investigate disease-mediated alterations in urothelial ATP release and detrusor contractility in the future, using bladders from animal diseased models and from humans. Acknowledgments. This study was supported by grants from the Singapore Ministry of Education (RG63/06 and RG83/07) and Nanyang Technological University (SUG15/07).
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