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Effects of Nitric Oxide on Detrusor Relaxation
0022-5347/96/1556-20$03.00/0
Vol. 155,2090-2093,June 1996 Printed in U S A .
THE JOLRNAL OF UROLOCY Copyright 0 1996 by AMERICAN UROLOCICAL ASS~CIATION, INC.
EFFECTS OF NITRIC OXIDE ON DETRUSOR RELAXATION BYUNG HA CHUNG, SEUNG KANG CHOI
AND
KI CHURL CHANG*
From the Department of Urology,Yonsei University College of Medicine, Seoul, and the Department of Pharmacology and Cardiovascular Research Institute, Gyeongsang National University College of Medicine, Chinju, Korea
ABSTRACT
Purpose: Recently we (1994)reported the photo-induced adequate nitric oxide (PIANO) system, in which an NO- or NO,-carrying molecule which has been photoactivated to release NO, could be exploited to investigate the role of NO in various smooth muscle functions. This study was designed to characterize the effect of nitric oxide (NO) exploiting PIANO on rat detrusor relaxation by isometric tension recording and measuring changes in cGMP content. Materials and Methods: Exposure to ultraviolet light was used (1 to 60 seconds) to evoke PIANO in the presence of streptozotocin, an NO-carrier, and Nu-nitro-L-arginine (L-NOARG),a n NO,-carrier. During relaxation the cyclic guanosine monophosphate (cGMP) content was measured by radioimmunoassay. Results: Rat detrusor strips were reversibly relaxed upon NO generation via PIANO. PFOgallol, an 0,generator, significantly (p <0.01) diminished PIANO-mediated relaxation. During PIANO-mediated relaxation, the tissue level of cyclic GMP significantly (p <0.05)increased over that of the control. Furthermore, methylene blue, a guanylate cyclase inhibitor, significantly (p <0.01)inhibited both the relaxation and the increase of cGMP. Conclusion: We concluded that rat detrusor muscle was capable of responding to NO, and these findings might lead to a treatment for bladder instability and detrusor hyperreflexia, by the use of intravesical instillation of NO donors. KEY WORDS: bladder, nitric oxide, rats, muscle relaxation The mechanism underlying relaxation of the bladder during filling is not fully understood. Recently it has been suggested that nitric oxide (NO) acts as a mediator of adaptive relaxation in the stomach to accommodate food or fluid. Theoretically, increased activity of NO-releasing inhibitory nerves could be a factor in keeping the bladder relaxed during the filling phase. The L-arginine/NO pathway has now been recognized in various organs and is an important and ubiquitous effector system in the regulation of a diverse set of physiological processes.13 Others have tested whether NO mediates relaxation in the bladder, but so far the results are conflicting.4-6 Furthermore, the mechanism underlying bladder relaxation during filling is not fully understood. We recently proposed the photo-induced adequate nitric oxide (PIANO) generating system as another investigational tool into the role of NO. It exploits the properties that the NOand NO,-canying molecules photoactivate to release NO.7-12 The PIANO-mediated relaxation study is effective in vascular smooth muscle, trachea, corpus cavernosum and gastrointestinal smooth muscle as well as in uterine smooth muscle.7-12 Thus, we investigated whether NO may play a role in bladder function via the PIANO-generating system. A preliminary account of this study was presented a t the American Urological Association Ninetieth Annual Meeting in an abstract.', MATERIALS AND METHODS
Drugs. Nw-nitro-L-arginine (L-NOARG), streptozotocin (STZ), carbachol chloride and pyrogallol (FVR) were purchased from Sigma Chemical Co. (St. Louis, Missouri). Methylene blue (MJ3) was from Fluka (Buchs, Switzerland). Muscle preparation. Male Sprague-Dawley rats (15to 18 weeks, 350 to 400 9.) were anesthetized with ketamine (0.75 Accepted for publication December 8, 1995. * Requests for reprints: Department of Pharmacology, College of Medicine, Gyeongsang National University, Chinju 660-280, Korea.
mg./kg., intraperitoneally) and xylazine (0.1mg./kg., intraperitoneally), and bled to death. Bladders were removed and cleaned of adhering fat and connective tissue, and strips (2 X 2 x 10 mm.) were prepared. Isometric tension study. Each strip was mounted in a 10 ml. water jacketed muscle chamber containing 37C modified Krebs-Ringer bicarbonate solution which was gassed with 95% 0,-5% CO, and had the following composition (mM.): NaCl (136.9),KC1 (5.4),MgC1, (1.01,NaHCO, (23.8),CaC1, (1.5), glucose (5.5) and EDTA (0.03).The tissues were equilibrated a t 1g. tension for more than 90 minutes, with washing at 20-minute intervals before drug addition. Isometric tension was recorded on a Grass physiograph (model 7E) with a force displacement transducer (FT-03, Grass Instruments, Quincy, Massachusetts). After a plateau of contraction was reached, tissues were exposed to ultraviolet (UV) light (1to 60 seconds) using a long wavelength UV lamp (366 nm., Mineralight U V GL 58,San Gabriel, California) in the presence or absence of test substances. In some experiments, PYR (10 pM., 100 pM.) or MB (100 pM.)was pretreated to ascertain how these probes affected PIANO-mediated relaxation. An ultraviolet lamp was faced a t a 5 cm. distance from the tissue preparations, since NO-mediated relaxation (photorelaxation) is reported to be proportional to the distance from the light source.13 Cyclic guanosine monophosphate (cGMP) determination. Cyclic GMP levels were measured in detrusor strips that had been equilibrated under tension and subjected to precontraction with carbachol(30 pM.). Strips were quickly frozen with the aid of brass clamps precooled in liquid nitrogen after being exposed to UV light for the indicated times (15, 30,60 and 180 seconds) in the presence of STZ (100 pM.1 and/or methylene blue (100 pM.).Samples were extracted and assayed for cGMP by radioimmunoassay as described.10 Statistics. Data are expressed as mean 5 SD. Differences between the 2 groups were determined by Student's t test and were considered significantly different if p <0.05.
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EFFECTS OF NO ON DETRUSOR RELAXATION
Drug concentrations were expressed as a final negative log,, molar concentration.
4
RESULTS
Relaxation mediated by PIANO. As shown in figure l,B 60carbachol (30 PMJ-induced tone of rat detrusor strips was not affected by UV light alone. However, after inclusion of control streptozotocin (STZ), an NO-carrying photosensitizer, it proSTZ-4 duced transient and reversible relaxation upon W irradiaL-NOARG -3.5 4a tion. In the presence of another photosensitizer, L-NOARG, carbachol-contracted rat detrusor strips were relaxed when P the tissue was exposed to UV light, even though the magnitude of relaxation was smaller than in those treated with STZ (fig. 2). In addition, the magnitude of relaxation was dependent on the exposure time to W light (figs. 1 and 2). The relaxation pattern vaned somewhat according to prepa0 ration; some strips had rebound contraction and others did not (fig. 1).The relaxant effect was usually smaller than that 0 20 40 60 observed in other smooth muscles, such as rat aorta and rabbit corpus cavernosum. Photoreleased NO is reported to UV exposure time (sec) be rapidly destroyed by 0, radicals and is protected by suFIG.2. Comparison of PIANO-mediated relaxation with different peroxide dismutase (SOD) in vascular smooth muscles.8 To confirm whether 0, could affect PIANO-mediated relaxation photosensitizers, streptozotocin [STZ (n = 2 0 ) and L-NOARG (n = 16)l on rat detrusor stri s Values represent mean z SD (’3) comin the rat bladder smooth muscle, pyrogallol (PYR), an 0, pared with carbachol-inguced contraction. generator, was used. As shown in fig. 1,PYR significantly (p <0.01) inhibited the STZ-potentiated photorelaxation. Methylene blue (MB), a guanylate cyclase inhibitor, also inhibited PIANO-mediated relaxation (fig. 1). Effects of PIANO-mediated relaxation on cyclic GMP levels. 10 l2 Ultraviolet irradiation alone had no influence on the level of cGMP. In the presence of STZ (100 pM.), however, the cyclic GMP content in the rat detrusor significantly (p c0.05)increased as the time of exposure to UV light (namely, the amount of NO generation) increased. For example, 30 and 60 second exposure to U V light in the presence of STZ increased cGMP 1.87 and 2.54 times over control (Walone). Moreover the increased accumulation of cGMP was significantly (p <0.01) inhibited by methylene blue (100 f l . ) (fig. 3).
: d‘
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A
FIG.3. Effects of STZ istreptozotocin, 100 FM.) and MB imethylene blue, 100 on c clic GMP concentrations in detrusor strips according to changes o t W irradiation. Values are means ipmol./ mg.) ? SD using 4 strips (for each test condition). Control: strips were submaximally contracted with carbachol, W 60: W irradiation for 60 seconds; STZ+UV15: UV irradiation for 15 seconds in presence of STZ; STZ+W30: W irradiation for 30 seconds in presence of STZ; STZ+W60: W irradiation for 60 seconds in presence of STZ; STZ+W180: W irradiation for 180 seconds in presence of STZ; STZ+MB+W15: W irradiation for 15 seconds in presence of STZ and MB, respectively. *, ** denote statistical significance for corresponding control a t p <0.05 and p 10.01,respectively.
a.)
10 rnin
DISCUSSION
0.5 g
Biological studies of “caged NO compounds”, that is, compounds that are stable in oxygen-containing solutions until photolysis by W irradiation causes them to release NO, have J-5.5 PYR recently been used as research tools for delivering Carbachol NO.7-13, 14.15 Streptozotocin has been reported to release NO FIG. 1. Representative tracings of PIANO-mediated relaxation in by us7-13 and others.IG.17 One of the most useful tools for rat detrusor smooth muscle employed in present experiment. Ultraviolet irradiation (60 seconds) without STZ (streptozotocin), NO- delivering NO at will is PIANO since it simply requires carrying molecule, had no influence on tone induced by carbachol. turning on and off the W light. Thus, PIANO-induced relaxHowever, in presence of STZ, W irradiation produced rapid, tran- ation appears to be a suitable means of studying the effect of sient and reversible time-dependent ( 1 to 60 seconds) relaxation. NO-mediated relaxation in various smooth muscles.i-12 The Each strip was submaximally precontracted with carbachol chloride. Note t h a t relaxation pattern varies somewhat according to tissues; present study clearly demonstrates that precontracted rat Some produced rebound contraction (A) but others did not (I?).Both detrusor smooth muscle relaxed to UV light only when cerP ogallol (PYR), an 0,generator, and methylene blue (MB), guan- tain amounts of NO- or NO,-carrying compounds were caged Y ate cyclase inhibitor, inhibited PIANO-mediated relaxation. Expoto release NO upon W light exposure. Of particular interest sure time to W light is indicated by dots on tracings, which re re the observation that the NO synthase inhibitor, sent seconds. // indicates interruution of tracing - for 30 (A) a n 1 2 0 was L-NOARG, relaxed detrusor smooth muscle in the presence minutes ( B ) ,respectively.
r
2092
EFFECTS OF NO ON DETRUSOR RELAXATION
of UV light, although the magnitude of relaxation was ing hyperpolarizing action may contribute in part to the smaller than that elicited by STZ. When test tubes contain- relaxation by PIANO in the rat bladder. Cyclic GMP producing the same concentrations of STZ (1 mM.) and L-NAME (1 tion was completely abolished by the treatment of MB and mM.) in solution were exposed to UV light (60seconds, 5 cm. even dropped below the basal level. The reason was not clear from the sample),nitrite accumulated in amounts of about 47 but this, in part, may be due to the high concentration of MB and 19 j N . , respectively.9 This may have been because NO2- we used, which could also have affected the basal level carrying molecules are decomposedby W light to release NO of &MI'. However, other studies showed that MB lowered from the molecules as demonstrated in rabbit corpus caver- cGMP levels below the basal level in other tissues.26 In connosum (CC)9-13 and in vascular smooth rnuscle.7.8.13 This clusion, transient, rapid and reversible relaxation was obsuggestion is further supported by our previous result which served in rat bladder strips exposed to PIANO. This relaxshowed that Nu-nitro-D-arginine methylester (D-NAME), an ation was accompanied by increases of cGMP and was inactive isomer of the NO synthase inhibitor, also had a inhibited in the presence of a guanylate cyclase inhibitor, similar effect as GNAME on PIANO-mediated relaxation in indicating that NO may serve a functional role in detrusor rabbit CC.9 Thus, PIANO-mediated relaxations differ from relaxation. We concluded that rat musclus detrusor is capable of rethose involved in the Larginine/NO (L-ARG/NO)pathway as reported in the rat aorta.7 Recently it has been reported that sponding to NO, which may play a role in relaxation of the decrease in NO production by NO synthase inhibitors may detrusor. These findings may lead to an approach to the increase the storage of photosensitive NO as a physiological treatment of bladder instability and detrusor hyperreflexia, compensation in vascular smooth muscle.18 However, the by the intravesical instillation of NO donors. detrusor smooth muscle, unlike vascular smooth muscle,19 Acknowledgments. The authors would like to thank Tim contains no photoactivatable store of NO, since UV irradia- Cornish for English language revision. tion alone can not relax it. Thus it seems unlikely that this finding is applicable to the rat detrusor smooth muscle. REFERENCES This finding suggested that rat detrusor smooth muscle does not contain a significant amount of endogenous photosensi1. Ignarro, L. J.: Biosynthesis and metabolism of endotheliumtive material. While numerous researchers have examined derived nitric oxide. Annu. Rev. Pharmacol. Toxicol., 30 535, 1990. the effects of cholinergic, adrenergic, purinergic and pepti2. Moncada, S.,Palmer, R. M. J. and Higgs, E. A.: Nitric oxide: dergic agents on bladder contraction,20 recent results impliphysiology, pathophysiology and pharmacology. Pharmacol. cate nitric oxide (NO) as an im-portant transmitter or mesRev., 43: 109,1991. senger molecule in auto-nomic neurotransmission.21-23 3. Stuehr, D.J. and Griffith, 0. W.: Mammalian nitric oxide synNonadrenergic noncholinergic (NANC)-nerve mediated rethases. Adv. Enzymol. Relat. Areas Mol. Biol., 65:287, 1992. laxation, involving the L-ARGNO pathway, has been dem4. Klarskov, P.: Non-cholinergic, non-adrenergic inhibitory nerve onstrated,4#5but not in detrusor smooth muscle.6 The present resDonses of bladder outlet smooth muscle in vitro. Br. J. experiment provided clues to the possible role of NO in uriUrol., 5 9 414, 1987. 5. James, M.J.. Birmingham. A. T. and Hill, S. J.: Partial medianary bladder function in the rat. In recent years it has been tion of nit& oxide o'i.the relaxation of human isolated detrusor demonstrated that NANC-nerve mediated relaxation in varstrips in response to electrical field stimulation. Br. J. Clin. ious smooth muscles is through an L-ARG/NO pathway, Pharmacol., 35:366, 1993. which is identical to NO.23.x We found that NADPH6. Persson, K and Andersson, K-E.: Nitric oxide and relaxation of diaphorase-positive nerve fibers were evident throughout the pig lower urinary tract. Br. J. Pharmacol., 106: 416, 1992. bladder but were generally more abundant near the bladder 7. Chang, K. C., Kim, Y. S. and Lee, S. Y.: Is the L-arginine/NO base (data not shown). If NO plays a physiologic role in the pathway involved in photorelaxation in rat aorta? Pharmacol. rat bladder, it could relax the detrusor smooth muscle, and Commun., 4 67,1993. the relaxation pattern, that is, the rate and duration of 8. Chang, K. C., Chong, W. S., Park, B. W., Seung, B. W., Chun, relaxation, should be similar to the pattern of authentic NO G. W., Lee, I. J. and Park, P. S.: NO- and NO,-carrying molecules potentiate photorelaxation in rat trachea and aorta. in other smooth muscles. As shown in the present study, Biochem. Biophys. Res. Commun., 191: 509, 1993. PIANO-mediated relaxation mimicks that induced by NANC 9. Chung, B. H. and Chang, K. C.: Photo-induced adequate nitric nerve stimulation in other tissues.23.24 Since NO is unstable oxide (PIANO)-mediated relaxation in isolated rabbit corpus and has a very short half life, the PIANO method may be a cavernosum. Gen. Pharmacol., 2 5 893, 1994. good method to study muscle kinetics with regard to NO. A 10. Lee, J. H.and Chang, K. C.: Different sensitivity to nitric oxide greater amount of nitrite, a stable NO product, accumulated of human pregnant and nonpregnant myometrial contractility. after exposure to high energy levels of light.9 This finding is Pharmacol. Commun., 5 147,1995. consistent with others in that release of NO depended on the 11. Chang, K. C.: Photo-induced adequate nitric oxide (PIANO) gendistance from the light source.13 Since the L-ARGNO/cyclic erating system as a useful tool for the investigation o f NOmediated responses. Br. J. Pharmacol., 1 1 4 272P, 1995. GMP system has an effective regulatory function in muscle relaxation,lA we further investigated whether the PIANO- 12. Chung, B. H., Choi, S. K. and Chang, K. C.: Effects of nitric oxide on detrusor relaxation. J. Urol., 153: 459A, 1995. generating system activated guanylate cyclase. Tissue cyclic GMP levels increased by PIANO, which was inhibited in the 13. Chen, X.and Gillis, C. N.: Methylene blue enhanced photorelaxation in aorta, pulmonary artery and corpus cavernosum. Biopresence of guanylate cyclase inhibitor. It seems likely, chem. Biophys. Res. Commun., 190: 559, 1993. therefore, that NO was responsible for the relaxation of rat 14. Pou, S.,Anderson, D. E., Surichamorn, W., Keaton, L. L. and detrusor by increasing cyclic GMP levels. But the increased Tod, M. L.: Biological studies of a nitroso compound that accumulation of cGMP did not always correlate well with releases nitric oxide upon illumination. Mol. Pharmacol., 46 muscle relaxation. For example, in the presence of photosen709, 1994. sitizers exposure of the tissues to UV light for as long as 30 15. Markings, L. R. and Tsien, R. Y.:Caged nitric oxide: stable organic molecules from which nitric oxide can be photoreseconds caused relaxation proportional to the increase of leased. J. Biol. Chem., 269 6282, 1994. cGMP in the tissues. Further exposure to UV light (60 to 180 seconds) increased tissue cGMP levels, but did not further 16. Turk, J., Corbett, J. A., Ramanadham, S., Bohrer, A. and McDaniel, M. L.: Biochemical evidence for nitric oxide formaincrease the magnitude of relaxation. The reason was not tion from streptozotocin in isolated pancreatic islets. Biochem. clear, but it seemed likely that other factor(s) may be inBiophys. Res. Commun., 191: 1458,1993. volved in PIANO-mediated relaxation. Hyperpolarizing ac- 17. Kwon, N. S.,Lee, S. H., Choi, C. S., Kho, T. and Lee, H. S.: Nitric tion is reported in W-induced relaxation in vascular smooth oxide from streptozotocin. F.A.S.E.B. J., 8 529, 1994. muscles;25 therefore we suggest that further studies concern- 18. Kubaszewski, E.,Peters, A., MacLain, S., Bohr, D. and Malinski,
EFFECTS OF NO ON DETRUSOR RELAXATION
T.: Light-activated release of nitric oxide from vascular smooth muscle of normotensive and hypertensive rats. Biochem. Biophysic. Res. Commun., 200: 213, 1994. 19. Ventrurini, C. M., Palmer, R. M. J. and Moncada, S.: Vascular smooth muscle contains a depletable store of a vasodilator which is light-activated and restored by donors of nitric oxide. J. Pharmacol. Exp. Ther., 266: 1497,1993. 20. de Groat, W.C. and Saum, W. R.: Sympathetic inhibition of the urinary bladder and of pelvic ganglionic transmission in the cat. J. Physiol., 220 297, 1972. 21. Buga, G. M. and Ignarro, L. J.: Electrical field stimulation causes endothelium-dependent nitric oxide-mediated relaxation of pulmonary artery. Am. J. Physiol., 262 H973, 1992. 22. Bush, P.A.,Aronson, W. J., Buga, G. M., Rajfer, J. and Ignarro, L. J.: Nitric oxide is a potent relaxant of human and rabbit
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corpus cavernosum. J. Urol., 141: 1650,1992. 23. Li, C. G.and Rand, M. J.: Nitric oxide and vasoactive intestinal polypeptide mediate nonadrenergic noncholinergic inhibitory transmission to smooth muscle of the rat gastric fundus. Eur. J. Pharmacol., 191: 303, 1990. 24. Andersson, K-E.: Pharmacology of lower urinary tract smooth muscles and penile erectile tissue. Pharmacol. Rev., 45 253,
1993. 25. Charpie, J. R., Peters, A. and Webb, R. C.: A photoactivable source of relaxing factor in genetic hypertension. Hypertension, 23: 894,1994. 26. Demerle-Pallardy, C., Lonchampt, M.-O., Chabrier, P.-E. and Braquet, P.: Absence of implication of Larginindnitric oxide pathway on neuronal cell injury induced by L-glutamate or hypoxia. Biochem. Biophys. Res. Commun., 181: 456, 1991.
Vol. 155,2090-2093,June 1996 Printed in U S A .
THE JOLRNAL OF UROLOCY Copyright 0 1996 by AMERICAN UROLOCICAL ASS~CIATION, INC.
EFFECTS OF NITRIC OXIDE ON DETRUSOR RELAXATION BYUNG HA CHUNG, SEUNG KANG CHOI
AND
KI CHURL CHANG*
From the Department of Urology,Yonsei University College of Medicine, Seoul, and the Department of Pharmacology and Cardiovascular Research Institute, Gyeongsang National University College of Medicine, Chinju, Korea
ABSTRACT
Purpose: Recently we (1994)reported the photo-induced adequate nitric oxide (PIANO) system, in which an NO- or NO,-carrying molecule which has been photoactivated to release NO, could be exploited to investigate the role of NO in various smooth muscle functions. This study was designed to characterize the effect of nitric oxide (NO) exploiting PIANO on rat detrusor relaxation by isometric tension recording and measuring changes in cGMP content. Materials and Methods: Exposure to ultraviolet light was used (1 to 60 seconds) to evoke PIANO in the presence of streptozotocin, an NO-carrier, and Nu-nitro-L-arginine (L-NOARG),a n NO,-carrier. During relaxation the cyclic guanosine monophosphate (cGMP) content was measured by radioimmunoassay. Results: Rat detrusor strips were reversibly relaxed upon NO generation via PIANO. PFOgallol, an 0,generator, significantly (p <0.01) diminished PIANO-mediated relaxation. During PIANO-mediated relaxation, the tissue level of cyclic GMP significantly (p <0.05)increased over that of the control. Furthermore, methylene blue, a guanylate cyclase inhibitor, significantly (p <0.01)inhibited both the relaxation and the increase of cGMP. Conclusion: We concluded that rat detrusor muscle was capable of responding to NO, and these findings might lead to a treatment for bladder instability and detrusor hyperreflexia, by the use of intravesical instillation of NO donors. KEY WORDS: bladder, nitric oxide, rats, muscle relaxation The mechanism underlying relaxation of the bladder during filling is not fully understood. Recently it has been suggested that nitric oxide (NO) acts as a mediator of adaptive relaxation in the stomach to accommodate food or fluid. Theoretically, increased activity of NO-releasing inhibitory nerves could be a factor in keeping the bladder relaxed during the filling phase. The L-arginine/NO pathway has now been recognized in various organs and is an important and ubiquitous effector system in the regulation of a diverse set of physiological processes.13 Others have tested whether NO mediates relaxation in the bladder, but so far the results are conflicting.4-6 Furthermore, the mechanism underlying bladder relaxation during filling is not fully understood. We recently proposed the photo-induced adequate nitric oxide (PIANO) generating system as another investigational tool into the role of NO. It exploits the properties that the NOand NO,-canying molecules photoactivate to release NO.7-12 The PIANO-mediated relaxation study is effective in vascular smooth muscle, trachea, corpus cavernosum and gastrointestinal smooth muscle as well as in uterine smooth muscle.7-12 Thus, we investigated whether NO may play a role in bladder function via the PIANO-generating system. A preliminary account of this study was presented a t the American Urological Association Ninetieth Annual Meeting in an abstract.', MATERIALS AND METHODS
Drugs. Nw-nitro-L-arginine (L-NOARG), streptozotocin (STZ), carbachol chloride and pyrogallol (FVR) were purchased from Sigma Chemical Co. (St. Louis, Missouri). Methylene blue (MJ3) was from Fluka (Buchs, Switzerland). Muscle preparation. Male Sprague-Dawley rats (15to 18 weeks, 350 to 400 9.) were anesthetized with ketamine (0.75 Accepted for publication December 8, 1995. * Requests for reprints: Department of Pharmacology, College of Medicine, Gyeongsang National University, Chinju 660-280, Korea.
mg./kg., intraperitoneally) and xylazine (0.1mg./kg., intraperitoneally), and bled to death. Bladders were removed and cleaned of adhering fat and connective tissue, and strips (2 X 2 x 10 mm.) were prepared. Isometric tension study. Each strip was mounted in a 10 ml. water jacketed muscle chamber containing 37C modified Krebs-Ringer bicarbonate solution which was gassed with 95% 0,-5% CO, and had the following composition (mM.): NaCl (136.9),KC1 (5.4),MgC1, (1.01,NaHCO, (23.8),CaC1, (1.5), glucose (5.5) and EDTA (0.03).The tissues were equilibrated a t 1g. tension for more than 90 minutes, with washing at 20-minute intervals before drug addition. Isometric tension was recorded on a Grass physiograph (model 7E) with a force displacement transducer (FT-03, Grass Instruments, Quincy, Massachusetts). After a plateau of contraction was reached, tissues were exposed to ultraviolet (UV) light (1to 60 seconds) using a long wavelength UV lamp (366 nm., Mineralight U V GL 58,San Gabriel, California) in the presence or absence of test substances. In some experiments, PYR (10 pM., 100 pM.) or MB (100 pM.)was pretreated to ascertain how these probes affected PIANO-mediated relaxation. An ultraviolet lamp was faced a t a 5 cm. distance from the tissue preparations, since NO-mediated relaxation (photorelaxation) is reported to be proportional to the distance from the light source.13 Cyclic guanosine monophosphate (cGMP) determination. Cyclic GMP levels were measured in detrusor strips that had been equilibrated under tension and subjected to precontraction with carbachol(30 pM.). Strips were quickly frozen with the aid of brass clamps precooled in liquid nitrogen after being exposed to UV light for the indicated times (15, 30,60 and 180 seconds) in the presence of STZ (100 pM.1 and/or methylene blue (100 pM.).Samples were extracted and assayed for cGMP by radioimmunoassay as described.10 Statistics. Data are expressed as mean 5 SD. Differences between the 2 groups were determined by Student's t test and were considered significantly different if p <0.05.
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EFFECTS OF NO ON DETRUSOR RELAXATION
Drug concentrations were expressed as a final negative log,, molar concentration.
4
RESULTS
Relaxation mediated by PIANO. As shown in figure l,B 60carbachol (30 PMJ-induced tone of rat detrusor strips was not affected by UV light alone. However, after inclusion of control streptozotocin (STZ), an NO-carrying photosensitizer, it proSTZ-4 duced transient and reversible relaxation upon W irradiaL-NOARG -3.5 4a tion. In the presence of another photosensitizer, L-NOARG, carbachol-contracted rat detrusor strips were relaxed when P the tissue was exposed to UV light, even though the magnitude of relaxation was smaller than in those treated with STZ (fig. 2). In addition, the magnitude of relaxation was dependent on the exposure time to W light (figs. 1 and 2). The relaxation pattern vaned somewhat according to prepa0 ration; some strips had rebound contraction and others did not (fig. 1).The relaxant effect was usually smaller than that 0 20 40 60 observed in other smooth muscles, such as rat aorta and rabbit corpus cavernosum. Photoreleased NO is reported to UV exposure time (sec) be rapidly destroyed by 0, radicals and is protected by suFIG.2. Comparison of PIANO-mediated relaxation with different peroxide dismutase (SOD) in vascular smooth muscles.8 To confirm whether 0, could affect PIANO-mediated relaxation photosensitizers, streptozotocin [STZ (n = 2 0 ) and L-NOARG (n = 16)l on rat detrusor stri s Values represent mean z SD (’3) comin the rat bladder smooth muscle, pyrogallol (PYR), an 0, pared with carbachol-inguced contraction. generator, was used. As shown in fig. 1,PYR significantly (p <0.01) inhibited the STZ-potentiated photorelaxation. Methylene blue (MB), a guanylate cyclase inhibitor, also inhibited PIANO-mediated relaxation (fig. 1). Effects of PIANO-mediated relaxation on cyclic GMP levels. 10 l2 Ultraviolet irradiation alone had no influence on the level of cGMP. In the presence of STZ (100 pM.), however, the cyclic GMP content in the rat detrusor significantly (p c0.05)increased as the time of exposure to UV light (namely, the amount of NO generation) increased. For example, 30 and 60 second exposure to U V light in the presence of STZ increased cGMP 1.87 and 2.54 times over control (Walone). Moreover the increased accumulation of cGMP was significantly (p <0.01) inhibited by methylene blue (100 f l . ) (fig. 3).
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i
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T
A
FIG.3. Effects of STZ istreptozotocin, 100 FM.) and MB imethylene blue, 100 on c clic GMP concentrations in detrusor strips according to changes o t W irradiation. Values are means ipmol./ mg.) ? SD using 4 strips (for each test condition). Control: strips were submaximally contracted with carbachol, W 60: W irradiation for 60 seconds; STZ+UV15: UV irradiation for 15 seconds in presence of STZ; STZ+W30: W irradiation for 30 seconds in presence of STZ; STZ+W60: W irradiation for 60 seconds in presence of STZ; STZ+W180: W irradiation for 180 seconds in presence of STZ; STZ+MB+W15: W irradiation for 15 seconds in presence of STZ and MB, respectively. *, ** denote statistical significance for corresponding control a t p <0.05 and p 10.01,respectively.
a.)
10 rnin
DISCUSSION
0.5 g
Biological studies of “caged NO compounds”, that is, compounds that are stable in oxygen-containing solutions until photolysis by W irradiation causes them to release NO, have J-5.5 PYR recently been used as research tools for delivering Carbachol NO.7-13, 14.15 Streptozotocin has been reported to release NO FIG. 1. Representative tracings of PIANO-mediated relaxation in by us7-13 and others.IG.17 One of the most useful tools for rat detrusor smooth muscle employed in present experiment. Ultraviolet irradiation (60 seconds) without STZ (streptozotocin), NO- delivering NO at will is PIANO since it simply requires carrying molecule, had no influence on tone induced by carbachol. turning on and off the W light. Thus, PIANO-induced relaxHowever, in presence of STZ, W irradiation produced rapid, tran- ation appears to be a suitable means of studying the effect of sient and reversible time-dependent ( 1 to 60 seconds) relaxation. NO-mediated relaxation in various smooth muscles.i-12 The Each strip was submaximally precontracted with carbachol chloride. Note t h a t relaxation pattern varies somewhat according to tissues; present study clearly demonstrates that precontracted rat Some produced rebound contraction (A) but others did not (I?).Both detrusor smooth muscle relaxed to UV light only when cerP ogallol (PYR), an 0,generator, and methylene blue (MB), guan- tain amounts of NO- or NO,-carrying compounds were caged Y ate cyclase inhibitor, inhibited PIANO-mediated relaxation. Expoto release NO upon W light exposure. Of particular interest sure time to W light is indicated by dots on tracings, which re re the observation that the NO synthase inhibitor, sent seconds. // indicates interruution of tracing - for 30 (A) a n 1 2 0 was L-NOARG, relaxed detrusor smooth muscle in the presence minutes ( B ) ,respectively.
r
2092
EFFECTS OF NO ON DETRUSOR RELAXATION
of UV light, although the magnitude of relaxation was ing hyperpolarizing action may contribute in part to the smaller than that elicited by STZ. When test tubes contain- relaxation by PIANO in the rat bladder. Cyclic GMP producing the same concentrations of STZ (1 mM.) and L-NAME (1 tion was completely abolished by the treatment of MB and mM.) in solution were exposed to UV light (60seconds, 5 cm. even dropped below the basal level. The reason was not clear from the sample),nitrite accumulated in amounts of about 47 but this, in part, may be due to the high concentration of MB and 19 j N . , respectively.9 This may have been because NO2- we used, which could also have affected the basal level carrying molecules are decomposedby W light to release NO of &MI'. However, other studies showed that MB lowered from the molecules as demonstrated in rabbit corpus caver- cGMP levels below the basal level in other tissues.26 In connosum (CC)9-13 and in vascular smooth rnuscle.7.8.13 This clusion, transient, rapid and reversible relaxation was obsuggestion is further supported by our previous result which served in rat bladder strips exposed to PIANO. This relaxshowed that Nu-nitro-D-arginine methylester (D-NAME), an ation was accompanied by increases of cGMP and was inactive isomer of the NO synthase inhibitor, also had a inhibited in the presence of a guanylate cyclase inhibitor, similar effect as GNAME on PIANO-mediated relaxation in indicating that NO may serve a functional role in detrusor rabbit CC.9 Thus, PIANO-mediated relaxations differ from relaxation. We concluded that rat musclus detrusor is capable of rethose involved in the Larginine/NO (L-ARG/NO)pathway as reported in the rat aorta.7 Recently it has been reported that sponding to NO, which may play a role in relaxation of the decrease in NO production by NO synthase inhibitors may detrusor. These findings may lead to an approach to the increase the storage of photosensitive NO as a physiological treatment of bladder instability and detrusor hyperreflexia, compensation in vascular smooth muscle.18 However, the by the intravesical instillation of NO donors. detrusor smooth muscle, unlike vascular smooth muscle,19 Acknowledgments. The authors would like to thank Tim contains no photoactivatable store of NO, since UV irradia- Cornish for English language revision. tion alone can not relax it. Thus it seems unlikely that this finding is applicable to the rat detrusor smooth muscle. REFERENCES This finding suggested that rat detrusor smooth muscle does not contain a significant amount of endogenous photosensi1. Ignarro, L. J.: Biosynthesis and metabolism of endotheliumtive material. While numerous researchers have examined derived nitric oxide. Annu. Rev. Pharmacol. Toxicol., 30 535, 1990. the effects of cholinergic, adrenergic, purinergic and pepti2. Moncada, S.,Palmer, R. M. J. and Higgs, E. A.: Nitric oxide: dergic agents on bladder contraction,20 recent results impliphysiology, pathophysiology and pharmacology. Pharmacol. cate nitric oxide (NO) as an im-portant transmitter or mesRev., 43: 109,1991. senger molecule in auto-nomic neurotransmission.21-23 3. Stuehr, D.J. and Griffith, 0. W.: Mammalian nitric oxide synNonadrenergic noncholinergic (NANC)-nerve mediated rethases. Adv. Enzymol. Relat. Areas Mol. Biol., 65:287, 1992. laxation, involving the L-ARGNO pathway, has been dem4. Klarskov, P.: Non-cholinergic, non-adrenergic inhibitory nerve onstrated,4#5but not in detrusor smooth muscle.6 The present resDonses of bladder outlet smooth muscle in vitro. Br. J. experiment provided clues to the possible role of NO in uriUrol., 5 9 414, 1987. 5. James, M.J.. Birmingham. A. T. and Hill, S. J.: Partial medianary bladder function in the rat. In recent years it has been tion of nit& oxide o'i.the relaxation of human isolated detrusor demonstrated that NANC-nerve mediated relaxation in varstrips in response to electrical field stimulation. Br. J. Clin. ious smooth muscles is through an L-ARG/NO pathway, Pharmacol., 35:366, 1993. which is identical to NO.23.x We found that NADPH6. Persson, K and Andersson, K-E.: Nitric oxide and relaxation of diaphorase-positive nerve fibers were evident throughout the pig lower urinary tract. Br. J. Pharmacol., 106: 416, 1992. bladder but were generally more abundant near the bladder 7. Chang, K. C., Kim, Y. S. and Lee, S. Y.: Is the L-arginine/NO base (data not shown). If NO plays a physiologic role in the pathway involved in photorelaxation in rat aorta? Pharmacol. rat bladder, it could relax the detrusor smooth muscle, and Commun., 4 67,1993. the relaxation pattern, that is, the rate and duration of 8. Chang, K. C., Chong, W. S., Park, B. W., Seung, B. W., Chun, relaxation, should be similar to the pattern of authentic NO G. W., Lee, I. J. and Park, P. S.: NO- and NO,-carrying molecules potentiate photorelaxation in rat trachea and aorta. in other smooth muscles. As shown in the present study, Biochem. Biophys. Res. Commun., 191: 509, 1993. PIANO-mediated relaxation mimicks that induced by NANC 9. Chung, B. H. and Chang, K. C.: Photo-induced adequate nitric nerve stimulation in other tissues.23.24 Since NO is unstable oxide (PIANO)-mediated relaxation in isolated rabbit corpus and has a very short half life, the PIANO method may be a cavernosum. Gen. Pharmacol., 2 5 893, 1994. good method to study muscle kinetics with regard to NO. A 10. Lee, J. H.and Chang, K. C.: Different sensitivity to nitric oxide greater amount of nitrite, a stable NO product, accumulated of human pregnant and nonpregnant myometrial contractility. after exposure to high energy levels of light.9 This finding is Pharmacol. Commun., 5 147,1995. consistent with others in that release of NO depended on the 11. Chang, K. C.: Photo-induced adequate nitric oxide (PIANO) gendistance from the light source.13 Since the L-ARGNO/cyclic erating system as a useful tool for the investigation o f NOmediated responses. Br. J. Pharmacol., 1 1 4 272P, 1995. GMP system has an effective regulatory function in muscle relaxation,lA we further investigated whether the PIANO- 12. Chung, B. H., Choi, S. K. and Chang, K. C.: Effects of nitric oxide on detrusor relaxation. J. Urol., 153: 459A, 1995. generating system activated guanylate cyclase. Tissue cyclic GMP levels increased by PIANO, which was inhibited in the 13. Chen, X.and Gillis, C. N.: Methylene blue enhanced photorelaxation in aorta, pulmonary artery and corpus cavernosum. Biopresence of guanylate cyclase inhibitor. It seems likely, chem. Biophys. Res. Commun., 190: 559, 1993. therefore, that NO was responsible for the relaxation of rat 14. Pou, S.,Anderson, D. E., Surichamorn, W., Keaton, L. L. and detrusor by increasing cyclic GMP levels. But the increased Tod, M. L.: Biological studies of a nitroso compound that accumulation of cGMP did not always correlate well with releases nitric oxide upon illumination. Mol. Pharmacol., 46 muscle relaxation. For example, in the presence of photosen709, 1994. sitizers exposure of the tissues to UV light for as long as 30 15. Markings, L. R. and Tsien, R. Y.:Caged nitric oxide: stable organic molecules from which nitric oxide can be photoreseconds caused relaxation proportional to the increase of leased. J. Biol. Chem., 269 6282, 1994. cGMP in the tissues. Further exposure to UV light (60 to 180 seconds) increased tissue cGMP levels, but did not further 16. Turk, J., Corbett, J. A., Ramanadham, S., Bohrer, A. and McDaniel, M. L.: Biochemical evidence for nitric oxide formaincrease the magnitude of relaxation. The reason was not tion from streptozotocin in isolated pancreatic islets. Biochem. clear, but it seemed likely that other factor(s) may be inBiophys. Res. Commun., 191: 1458,1993. volved in PIANO-mediated relaxation. Hyperpolarizing ac- 17. Kwon, N. S.,Lee, S. H., Choi, C. S., Kho, T. and Lee, H. S.: Nitric tion is reported in W-induced relaxation in vascular smooth oxide from streptozotocin. F.A.S.E.B. J., 8 529, 1994. muscles;25 therefore we suggest that further studies concern- 18. Kubaszewski, E.,Peters, A., MacLain, S., Bohr, D. and Malinski,
EFFECTS OF NO ON DETRUSOR RELAXATION
T.: Light-activated release of nitric oxide from vascular smooth muscle of normotensive and hypertensive rats. Biochem. Biophysic. Res. Commun., 200: 213, 1994. 19. Ventrurini, C. M., Palmer, R. M. J. and Moncada, S.: Vascular smooth muscle contains a depletable store of a vasodilator which is light-activated and restored by donors of nitric oxide. J. Pharmacol. Exp. Ther., 266: 1497,1993. 20. de Groat, W.C. and Saum, W. R.: Sympathetic inhibition of the urinary bladder and of pelvic ganglionic transmission in the cat. J. Physiol., 220 297, 1972. 21. Buga, G. M. and Ignarro, L. J.: Electrical field stimulation causes endothelium-dependent nitric oxide-mediated relaxation of pulmonary artery. Am. J. Physiol., 262 H973, 1992. 22. Bush, P.A.,Aronson, W. J., Buga, G. M., Rajfer, J. and Ignarro, L. J.: Nitric oxide is a potent relaxant of human and rabbit
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corpus cavernosum. J. Urol., 141: 1650,1992. 23. Li, C. G.and Rand, M. J.: Nitric oxide and vasoactive intestinal polypeptide mediate nonadrenergic noncholinergic inhibitory transmission to smooth muscle of the rat gastric fundus. Eur. J. Pharmacol., 191: 303, 1990. 24. Andersson, K-E.: Pharmacology of lower urinary tract smooth muscles and penile erectile tissue. Pharmacol. Rev., 45 253,
1993. 25. Charpie, J. R., Peters, A. and Webb, R. C.: A photoactivable source of relaxing factor in genetic hypertension. Hypertension, 23: 894,1994. 26. Demerle-Pallardy, C., Lonchampt, M.-O., Chabrier, P.-E. and Braquet, P.: Absence of implication of Larginindnitric oxide pathway on neuronal cell injury induced by L-glutamate or hypoxia. Biochem. Biophys. Res. Commun., 181: 456, 1991.