Altered Nitric Oxide Synthase, Arginase and Ornithine Decarboxylase Activities, and Polyamine Synthesis in Response to Ischemia of the Rabbit Detrusor

Altered Nitric Oxide Synthase, Arginase and Ornithine Decarboxylase Activities, and Polyamine Synthesis in Response to Ischemia of the Rabbit Detrusor Keizo Kawano,* Hitoshi Masuda,† Masataka Yano, Kazunori Kihara, Akiko Sugimoto and Hiroshi Azuma From the Departments of Urology and Reproductive Medicine and Biosystem Regulation, Institute of Biomaterials and Bioengineering, Graduate School, Tokyo Medical and Dental University, Tokyo, Japan

Purpose: Little is known about L-arginine catabolism following ischemia in the bladder. We examined the changes in nitric oxide synthase, arginase and ornithine decarboxylase activity, polyamine biosynthesis and the ability to produce nitric oxide following ischemia of the rabbit bladder. Materials and Methods: Bladder ischemia was created by ligation of a unilateral bladder artery. At various time points, that is 1, 4, 8, 24, 48 and 72 hours following ligation, the bladders were excised and harvested for determinations. Results: Constitutive nitric oxide synthase, inducible nitric oxide synthase arginase and ornithine decarboxylase activities increased with time, peaking at 48 hours without significant differences between the ligated and nonligated sides in the whole layer. Arginase and ornithine decarboxylase increased mainly in the muscularis following ischemia. Also, putrescine in the muscularis was significantly higher than in the mucosa 48 hours following ischemia. Baseline nitrite/nitrate production in the whole detrusor on the ligated side at 24 hours was significantly lower than that in the normal detrusor. However, nor-hydroxyarginine as an arginase inhibitor and L-arginine increased nitrite/nitrate production in the ischemic detrusor without changing in the normal detrusor. This increasing effect of nor-hydroxyarginine was abolished by nitroarginine methylester as a nitric oxide synthase inhibitor. Conclusions: Enzymes related to L-arginine catabolism were involved in the early events of ischemic bladder. Arginase may have 2 independent roles, that is 1) activation of arginase/ornithine decarboxylase/polyamines pathways in the muscle injury and remodeling following ischemia, and 2) endogenous negative regulation of nitric oxide production by limiting the L-arginine substrate for nitric oxide synthase. Key Words: bladder, ischemia, rabbits, arginine, enzymes

be associated with an inflammatory response in which several mediators are released or activated, such as inflammatory cytokines and adhesion molecules.4 Cytokines have been reported to activate the expression of inflammation related genes, iNOS and cyclooxygenase-2.5 Recent studies show that iNOS mRNA, protein and activity are up-regulated after bladder outlet obstruction.6 However, little is known about the NOS isoform changes related to bladder ischemia. L-arginine is a substrate not only for NOS, but also for arginase, which catalyzes the conversion of L-arginine to L-ornithine and urea. Therefore, L-arginine catabolism via the arginase pathway can act as an endogenous, negative regulation system for overall NO production.7 Arginase and iNOS activities are regulated reciprocally in macrophages by cytokines, which may guarantee efficient NO production.8 Arginase also has an important role in providing L-ornithine for the synthesis of polyamines controlling cell proliferation and differentiation. ODC is the rate limiting enzyme for the conversion of L-ornithine to putrescine, which is further converted to spermidine by spermidine synthase. It has been reported that cerebral ischemia produces a significant increase in ODC activity and putrescine levels.9 We investigated whether the activities of NOS, arginase and ODC polyamine levels change following ischemia and

linical and basic studies suggest that ischemia may have a significant role in bladder dysfunction. Arterial occlusion caused by pelvic surgery and arteriosclerosis as well as arterial compression due to outlet obstruction have been shown to cause significant bladder ischemia in the rabbit model, leading to contractile dysfunction, an increase in bladder mass and a marked decrease in bladder compliance and capacity.1 Recently NO was reported to be involved in the pathogenesis of bladder dysfunction. NO is synthesized from Larginine by NOS and 3 distinct isoforms of NOS have been identified, including iNOS, eNOS and nNOS. iNOS has been reported to produce a large amount of NO in response to inflammatory cytokines.2 Expression of iNOS has been described in the bladder urothelium, smooth muscle and stromal cells as well as in inflammatory cells during urinary tract infection.3 Ischemic brain injury has been reported to

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Submitted for publication July 22, 2005. Study complied with the Animal Welfare Regulation of Tokyo Medical and Dental University. * Correspondence: Department of Urology, Tokyo Medical and Dental University, 1-5-45 Yushima, Bynkyo ward, Tokyo 113-8519, Japan (e-mail: [email protected]). † Financial interest and/or other relationship with Indevus and Pfizer.

0022-5347/06/1761-0387/0 THE JOURNAL OF UROLOGY® Copyright © 2006 by AMERICAN UROLOGICAL ASSOCIATION

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Vol. 176, 387-393, July 2006 Printed in U.S.A. DOI:10.1016/S0022-5347(06)00515-5

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TABLE 1. cNOS and iNOS activity before and after unilateral ligation of ipsilateral mucosa, muscularis and whole layer Mean Pmol/Mg Protein/Min ⫾ SEM

Ca2⫹ dependent cNOS: Whole layer Mucosa Muscularis Ca2⫹ independent iNOS: Whole layer Mucosa Muscularis

Control

Max*

7.52 ⫾ 0.76 11.3 ⫾ 0.91 2.50 ⫾ 0.31

26.7 ⫾ 1.41 30.9 ⫾ 2.58 11.2 ⫾ 0.85

2.04 ⫾ 0.14 2.12 ⫾ 0.16 1.70 ⫾ 0.15

11.7 ⫾ 2.11 12.9 ⫾ 2.05 8.95 ⫾ 1.22

* At 48 hours on ligated side.

whether NO production is modulated by arginase in the ischemic bladder. Also, we have previously reported the different distributions of NOS activity in the mucosa and the muscularis of the rabbit detrusor.10 Therefore, we investigated the distribution of NOS, arginase and ODC polyamine levels in the mucosa and muscularis as well as different responses to ischemia. MATERIALS AND METHODS This study complied with the Animal Welfare Regulation of Tokyo Medical and Dental University. Experimental Protocol A total of 70 Japanese white male rabbits weighing approximately 3.0 kg were anesthetized by intravenous injection of sodium pentobarbital (25 mg/kg). Unilateral ischemia was created by surgically tying the major artery entering the bladder on the right side with 2-zero silk via an abdominal midline incision. Control rabbits underwent sham surgery without tying. The incision was closed and the anesthetized rabbit was observed until fully awake with normal movement. The rabbits were sacrificed by exsanguination 1, 4, 8, 24, 48 and 72 hours after unilateral ischemia, respectively, under pentobarbital anesthesia. The urinary tract was removed en bloc, maintained in a Petri dish containing ice-cold modified Krebs solution and dissected free of adipose and connective tissues. The bladder body and base were separated at the level of the ureteral orifices and the bladder body was divided into right (ligated side) and left (nonligated

side) halves. Under a dissecting microscope the mucosa of the bladder body was stripped away from the underlying muscularis, as reported by Levin et al.1 The mucosa consists of the urothelium, lamina propria, and vascular and connective tissues. All tissues were frozen immediately after dissection and stored at – 80C until analysis. Protein Preparation Tissue was homogenized at maximum speed for 15 seconds each 4 times to a 25% homogenate in buffer (1 ml/100 mg tissue), consisting of 320 mM sucrose, 10 mM HEPES, 0.1 mM ethylenediaminetetraacetic acid, 1 ␮M pepstatin and 1 ␮M leupeptin (pH 7.2). The homogenate was centrifuged at 10,000 ⫻ gravity for 20 minutes at 4C and the supernatant was decanted from the pellet. NOS Activity NOS activity was measured by determining the conversion of [3H]-L-arginine to [3H]-L-citrulline according to a previously described method.10 To measure iNOS activity (calcium independent NOS activity) CaCl2 was omitted and replaced with ethylenediaminetetraacetic acid (20 mM). Enzyme activity is expressed in pmol citrulline per gm protein per minute. Arginase Activity Arginase activity was measured by determining the conversion of L-[guanidido-14C]-arginine to [14C]-urea according to a previously described method.11 Arginase activity is expressed in pmol urea per mg protein per 60 minutes. ODC Activity ODC activity was determined as described by Dempsey et al.12 Briefly, ODC was determined by the amount of 14CO2 derived from 0.5 ␮Ci L-[14C]-ornithine. ODC activity is expressed in pmol 14CO2 per mg protein per 60 minutes. Tissue Polyamine Determination by Thin Layer Chromatography Polyamines were quantified as described by Seiler13 with minor modifications. Briefly, bladder tissue was homogenized in 0.3 M perchloric acid. Homogenates were centrifuged at 12,000 ⫻ gravity for 20 minutes and the

FIG. 1. Time course of Ca dependent (A) and Ca independent (B) NOS activity in whole layer bladder tissue after unilateral bladder artery ligation. Data represent mean ⫾ SEM (vertical bars) of 6 measurements in different animals. Single asterisk indicates p ⬍0.05 vs control. Double asterisks indicate p ⬍0.01.

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FIG. 2. Time course of Ca dependent (A) and Ca independent (B) NOS activity in mucosa and muscularis on ligated side of rabbit detrusor. Data represent mean ⫾ SEM (vertical bars) of 6 measurements in different animals. Single asterisk indicates p ⬍0.05 vs control. Double asterisks indicate p ⬍0.01. Dagger indicates ANOVA p ⬍0.01 vs muscularis.

supernatant was dansylated with 0.2 M Dns-Cl (5-dimethylaminonaphthalane-1-sulfonylchloride), 1 M Na2CO3 and acetone. Silica gel thin layer plates were developed with cyclohexane-ethyl acetate (1:1) on the first run, followed by a second run with cyclohexane-diethyl ether (3:2) in the same direction. The separated components were visualized under ultraviolet light (254 and 360 nm) using a Model UV 5L-58 Mineralight® Lamp. The separated spots were cut off with scissors and soaked in methanol. After centrifugation the fluorescence intensity of the supernatant was estimated using a Versa FluorTM spectrofluorometer (␭ex 365 nm and ␭em 510 nm). Polyamine levels are expressed in pmol per gm wet weight. NO Production in Cultured Detrusor Specimens At 24 hours the whole detrusor tissue from the ligated side was dissected into pieces approximately 200 mg each and separately incubated at 37C for 8 hours in modified Krebs solution to determine the tissue nitrite/nitrate level as an index of NO production. Tissues were incubated in the presence or absence of L-arginine (3, 10 or 25 mM), nor-NOHA (1 mM) or L-NAME (1 mM). Cultured tissue was homogenized in Krebs solution. The homogenate was centrifuged in Durapore™ centrifugal filter units to remove protein at 5,000 ⫻ gravity for 60 minutes at 4C and the supernatant was decanted from the pellet. Total nitrate in the supernatant was assayed using an NO2/NO3 assay kit-F2 (Dojindo Molecular Technologies, Gaithersburg, Maryland), in which samples were derivatized with 2, 3-diaminonaphthalene and applied to a fluorophotometer (␭ex 365 nm and ␭em 450 nm). Results were compared with those in control detrusor. Statistical Analysis Results are shown as the mean ⫾ SEM. Deviations from the mean regarding time to response curves were statistically analyzed by factorial 2-way ANOVA. Student 2-tailed t test was used for paired and unpaired data with p ⬍0.05 considered statistically significant. RESULTS Distribution of NOS Activities of the Detrusor and Different Responses to Ischemia by Mucosa and Muscularis In the control whole layer cNOS activity was much higher than iNOS activity (table 1, p ⬍0.05). Whole detrusor cNOS

and iNOS activities significantly increased with time (p ⬍0.05 and ⬍0.01, respectively), peaking at 48 hours not only on the ligated, but also on the nonligated side. There were no significant differences in NOS activities between the 2 sides at any time. On each side time to onset of the increase in iNOS activity was delayed compared with the increase in cNOS activity (fig. 1). In the mucosa cNOS activity was about 5 times higher than that in the muscularis in the control. On the ligated side cNOS activity in the mucosa and muscularis increased with a time course similar to that of the activity of the whole layer following ischemia (fig. 2, A). However, cNOS activity in the mucosa was much higher than that in the muscularis at any time. On the other hand, iNOS activity in the detrusor on the ligated side significantly increased in the mucosa and muscularis to the same extent (fig. 2, B). Distribution of Arginase and ODC Activities of the Detrusor, and Different Responses to Ischemia by Mucosa and Muscularis There were no significant differences in baseline arginase and ODC activities between the mucosa and muscularis (table 2). Arginase and ODC activities of the whole layer on the ligated and nonligated sides increased with time to the same extent, peaking at 48 hours. There were no significant differences in arginase and ODC activities between the 2 sides at any time (figs. 3, A and 4, A). On the ligated side arginase and ODC activities in the mucosa and muscularis increased with time. However, the increasing effects of arginase and ODC activities in the

TABLE 2. Arginase and ODC activity before and after unilateral ligation of ipsilateral mucosa, muscularis and whole layer Mean Pmol/Mg Protein/Hr ⫾ SEM

Arginase: Whole layer Mucosa Muscularis ODC: Whole layer Mucosa Muscularis * At 48 hours on ligated side.

Control

Max*

122 ⫾ 28.3 89.0 ⫾ 14.5 94.0 ⫾ 10.5

426 ⫾ 54.6 374 ⫾ 22.4 997 ⫾ 69.8

189 ⫾ 6.14 67.3 ⫾ 4.98 234 ⫾ 11.6

1,217 ⫾ 114 792 ⫾ 25.0 1,864 ⫾ 98.2

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ALTERATION IN L-ARGININE CATABOLISM IN ISCHEMIC RABBIT DETRUSOR

FIG. 3. Time course of arginase activity in whole layer bladder tissue after bladder artery ligation (A), and in mucosa and muscularis on ligated side of rabbit detrusor (B). Data represent mean ⫾ SEM (vertical bars) of 6 measurements in different animals. Single asterisk indicates p ⬍0.05 vs control. Double asterisks indicate p ⬍0.01. Daggers indicate ANOVA p ⬍0.001 vs mucosa.

muscularis were much higher than those in the mucosa at 48 and 72 hours (figs. 3, B and 4, B). Production of Nitrite/Nitrate by Ischemic Detrusor Tissue In the ischemic detrusor L-arginine increased the tissue nitrite/nitrate level in a concentration dependent manner (fig. 5, A). Also, the tissue nitrite/nitrate level was significantly increased by nor-NOHA as an inhibitor of arginase, of which an increasing effect was inhibited by 1 mM L-NAME. The baseline nitrite/nitrate level in normal bladder tissue was significantly higher than that in ischemic bladder tissue (1,082 ⫾ 131 vs 689 ⫾ 184 pmol nitrate per mg protein, p ⬍0.05). It remained unaffected in the presence of L-arginine (3, 10 and 25 mM) and nor-NOHA (1 mM) (fig. 5, B). Polyamine Levels in Ischemic Detrusor Tissue Putrescine levels in the mucosa and muscularis of the detrusor on the ligated side gradually increased with time (fig. 6). At 48 hours putrescine in the muscularis was significantly higher than in the mucosa. Spermidine did not change with time, whereas the spermine level was slightly but significantly decreased 24 to 48 hours later.

Changes in polyamine levels on the nonligated side of the bladder were quite similar to those observed on the ligated side (data not shown). DISCUSSION In the current study cNOS (eNOS plus nNOS), iNOS, arginase and ODC activities in the detrusor increased with time, peaking at 48 hours on the ligated and nonligated sides. There were no significant differences in these activities between the 2 sides. These findings suggest that unilateral ligation activates the enzymes related to L-arginine catabolism in the whole bladder. Saito et al observed that bladder blood flow after unilateral ligation was significantly decreased on the nonligated side as well as on the ligated side immediately and the smooth muscle layer was injured on each side 2 weeks after ischemia morphologically.14 Also, unilateral ligation of the vesical artery was reported to produce similar molecular responses on each side15 and decrease the response to bethanechol and field stimulation on each side (ligated ⬎ nonligated).16 It has been reported that there are many anatomically close communications of blood vessels over the detrusor.15

FIG. 4. Time course of ODC activity in bladder tissue after bladder artery ligation (A) and in mucosa and muscularis on ligated side of rabbit detrusor (B). Data represent mean ⫾ SEM (vertical bars) of 6 measurements in different animals. Single asterisk indicates p ⬍0.05 vs control. Double asterisks indicate p ⬍0.01. Daggers indicate ANOVA p ⬍0.001 vs mucosa.

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FIG. 5. Effect in 8 preparations of arginase inhibition by 10-3 mol/l of arginase inhibitor nor-NOHA on L-arginine (L-Arg) responses in rabbit bladder on ligated side (A). After 24 hours of unilateral ligation ipsilateral bladder tissue was dissected into 200 mg pieces and separately incubated at 37C for 8 hours in modified Krebs solution as control, in 3, 10 and 25 mM L-arginine, in 1 mM nor-NOHA (nor NOHA) and in 1 mM of NOS inhibitor L-NAME. Single asterisk indicates p ⬍0.05 vs control. Five preparations of unoperated whole layer bladder tissue were separately incubated under same conditions (B). Double asterisks indicate p ⬍0.05 vs operated control.

Therefore, it is likely that unilateral ligation causes an ischemic change over the whole detrusor. Unilateral ligation seemed to be a suitable experimental model for partial ischemia, which is often observed in pelvic surgery, urinary retention and vascular diseases. In the current study we did not investigate the role of each NOS isoform in response to detrusor ischemia. Gene knockout studies have determined that NO derived from eNOS is beneficial for acute ischemic stroke, which may be due in part to the preservation of blood flow.17 In contrast, NO produced by nNOS and iNOS can be neurotoxic, which probably occurs through NO induced formation of peroxynitrite and toxic free radicals.17 Increased cNOS activity with time seems to be due to increased blood flow in response to bladder ischemia. iNOS up-regulation has been reported to be involved in detrusor injury by ischemia-reperfusion.18 Further investigations

should be performed to clarify the role of each NOS isoform using the selective NOS inhibitors in this study. In our tissue culture study baseline NO (nitrite/nitrate) production was significantly lower in the ischemic detrusor than in the control irrespective of the up-regulation of cNOS and iNOS activity. However, the addition of excess L-arginine restored NO production in the ischemic detrusor without changing that in the control, suggesting that NO production in the ischemic state depends on the extracellular L-arginine concentration. It has been reported that iNOS activity depends strictly on the presence of extracellular arginine. When iNOS is induced and the high output pathway of NOS is activated, the L-arginine concentration and availability may become rate limiting for NO production. Our Krebs tissue culture medium did not contain L-arginine. Therefore, decreased baseline NO production in the ischemic bladder may not necessarily

FIG. 6. Levels of polyamines putrescine (A), spermidine (B) and spermine (C) in mucosa and muscularis bladder tissue on ligated side 24 to 72 hours after right bladder artery ligation in 5 preparations each. Single asterisk indicates p ⬍0.05. Double asterisks indicate p ⬍0.01 vs control.

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ALTERATION IN L-ARGININE CATABOLISM IN ISCHEMIC RABBIT DETRUSOR

reflect the in vivo state. On the other hand, nor-NOHA as an arginase inhibitor increased NO production, of which increasing effects were abolished by L-NAME as a NOS inhibitor. Arginase metabolizes L-arginine to urea and L-ornithine through the urea cycle. These findings suggest that the inhibition of enhanced arginase activity in the ischemic detrusor increased L-arginine availability to NOS (maybe iNOS), thereby increasing NO production. The role of arginase in modulating NO production is not limited to the ischemic bladder. Arginase has been reported to be up-regulated by aging and in diabetes mellitus in human and rabbit corpus cavernosum, and it is associated with erectile dysfunction.11 Arginase inhibitors have been reported to restore the impaired cavernous relaxation associated with aging and diabetes mellitus.11 Arginase also promotes the synthesis of L-ornithine. ODC catalyzes the formation of putrescine and CO2 from L-ornithine and it is the key regulatory enzyme in polyamine biosynthesis.7 The polyamines putrescine, spermidine and spermine are classically known to be important mediators of cell growth and division,9 in view of their capability to directly bind to DNA and modulate DNAprotein interactions. In this study arginase and ODC activities increased mainly in the muscularis following ischemia and putrescine in the muscularis was significantly higher than that in the mucosa 48 hours following ischemia. It has been reported that ODC activity and putrescine increase after brain ischemia in proportion to the extent of bloodbrain barrier dysfunction9 and difluoromethylornithine as an inhibitor of ODC protects blood-brain barrier dysfunction following brain ischemia. Hasegawa et al observed that myocardial polyamines were increased in the myocardial infarction of rats on day 3.19 These findings suggested that an increased arginase/ODC/polyamine pathway following ischemia may be involved in injury and remodeling in the ischemic region. Bladder ischemia has been shown to produce low compliance and hyperreflexia with decreased detrusor contractility.16 On in vivo cystometry unilateral ischemia caused a marked decrease in bladder compliance and capacity.16 Pretreatment with nonselective NOS inhibitors has been reported to prevent the decreased detrusor contractility caused by ischemia-reperfusion.20 Treatments with selective NOS inhibitors, arginase or ODC inhibitors before and during ischemia should be performed to investigate the links between these biochemical studies, and functional in vitro and in vivo findings.

Abbreviations and Acronyms cNOS eNOS iNOS L-NAME nNOS NO nor-NOHA NOS ODC

constitutive NOS endothelial NOS inducible NOS nitroarginine methylester neuronal NOS nitric oxide nor-hydroxyarginine NO synthase ornithine decarboxylase

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2.

3.

4.

5.

6.

7. 8.

9.

10.

CONCLUSIONS The enzymes involved in L-arginine catabolism in the detrusor were significantly activated with the concomitant accumulation of putrescine in response to ischemia. Arginase may have 2 independent roles in the early events of ischemic bladder, that is 1) endogenous negative regulation of overall NO production by limiting arginine substrate for NOS and 2) activation of arginase/ODC/ polyamines pathways, which may have a role in muscle injury and remodeling with ischemia.

⫽ ⫽ ⫽ ⫽ ⫽ ⫽ ⫽ ⫽ ⫽

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ALTERATION IN L-ARGININE CATABOLISM IN ISCHEMIC RABBIT DETRUSOR 14. Saito, M., Yokoi, K., Ohmura, M. and Kondo, A.: Effects of ligation of the internal iliac artery on blood flow to the bladder and detrusor function in rat. Int Urol Nephrol, 30: 283, 1998 15. Chen, M. W., Buttyan, R. and Levin, R. M.: Genetic and cellular response to unilateral ischemia of the rabbit urinary bladder. J Urol, 155: 732, 1996 16. Lin, A. T., Wein, A. J., Gill, H. S. and Levin, R. M.: Functional effect of chronic ischemia on the rabbit urinary bladder. Neurourol Urodyn, 7: 1, 1988 17. Moro, M. A., Cardenas, A., Hurtado, O., Leza, J. C. and Lizasoain, I.: Role of nitric oxide after brain ischemia. Cell Calcium, 36: 265, 2004

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Saito, M. and Miyagawa, I.: Direct detection of nitric oxide in rat urinary bladder during ischemia-reperfusion. J Urol, 162: 1490, 1999 19. Hasegawa, S., Nakano, M., Hamana, K., Taniguchi, Y., Iwasaki, T., Kanda, T. et al: Decrease in myocardial polyamine concentration in rats with myocardial infarction. Life Sci, 60: 1643, 1997 20. Saito, M., Wada, K., Kamisaki, Y. and Miyagawa, I.: Effect of ischemia-reperfusion on contractile function of rat urinary bladder: possible role of nitric oxide. Life Sci, 62: 149, 1998