Roles of Accumulated Endogenous Nitric Oxide Synthase Inhibitors and Decreased Nitric Oxide Synthase Activity for Impaired Trigonal Relaxation With Ischemia

0022-5347/03/1704-1415/0 THE JOURNAL OF UROLOGY® Copyright © 2003 by AMERICAN UROLOGICAL ASSOCIATION

Vol. 170, 1415–1420, October 2003 Printed in U.S.A.

DOI: 10.1097/01.ju.0000075098.36442.62

ROLES OF ACCUMULATED ENDOGENOUS NITRIC OXIDE SYNTHASE INHIBITORS AND DECREASED NITRIC OXIDE SYNTHASE ACTIVITY FOR IMPAIRED TRIGONAL RELAXATION WITH ISCHEMIA HITOSHI MASUDA,* MASATAKA YANO, YASUYUKI SAKAI, KAZUNORI KIHARA, MORITAKA GOTO 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

ABSTRACT

Purpose: We examined whether endogenous nitric oxide (NO) synthase (NOS) inhibitors are involved in the impaired trigonal relaxation with ischemia in rabbits. Materials and Methods: Rabbits were divided into control and ischemia groups. Two weeks after partial vessel occlusion strips of trigone and detrusor were processed to determine endogenous methylarginines and L-arginine by automated high performance liquid chromatography. We also compared NOS activity and NO mediated functional responses to electrical field stimulation between 2 groups. Results: Neurogenic and NO but not sodium nitroprusside induced mediated relaxation in the trigone were significantly impaired following ischemia. Ca2⫹ dependent NOS activity, and baseline and stimulated cyclic guanosine monophosphate production with electrical field stimulation were significantly decreased following ischemia. The contents of L-NMMA (NGmonomethyl-L-arginine) and asymmetrical ADMA (NG, NG–dimethyl-L-arginine) but not L-arginine or symmetrical SDMA (NG, N⬘G–dimethyl-L-arginine) were increased in the trigone following ischemia. Authentic L-NMMA and ADMA but not SDMA inhibited neurogenic relaxations in a concentration dependent manner without affecting the relaxation produced by sodium nitroprusside in control tissue. Excess L-arginine abolished L-NMMA and ADMA inhibition. Conclusions: These results suggest that impaired NO mediated trigonal relaxation following ischemia is closely related to decreased NOS activity and the increased accumulation of L-NMMA and ADMA. KEY WORDS: bladder, rabbits, nitric-oxide synthase, ischemia, relaxation

Vallance et al obtained evidence that L-NMMA (NGmonomethyl-L-arginine) and ADMA (NG, NG–dimethyl-Larginine) have a role as endogenous inhibitors for nitric oxide (NO) synthase (NOS).1 Increased methylarginines within cells and tissues may be a mechanism for NOS regulation. Recently it has been reported that the accumulation of endogenous NOS inhibitors in regenerated endothelial cells is associated with decreased NO production and the occurrence of intimal hyperplasia following endothelial denudation of the rabbit carotid artery.2 The concentration of these inhibitors was increased in plasma with peripheral arterial occlusive disease3 and in endothelial cells with diabetes mellitus.4 Moreover, in the noncardiovascular field we reported that the 3 methylarginines L-NMMA, ADMA and SDMA (NG, N⬘G–dimethyl-L-arginine) as well as NOS are localized throughout the rabbit lower urinary tract, and L-NMMA and ADMA but not SDMA inhibited Ca2⫹ dependent NOS activity and neurogenic NO mediated relaxation in the trigone and proximal urethra.5 Also, relaxation produced in isolated pig6 and sheep7 trigonal strips has been reported to be decreased by NOS inhibitors. Recently we reported that impaired NO mediated urethral relaxation due to ischemia is closely related to the accumulation of L-NMMA and ADMA in urethral tissue.8 Therefore, we investigated the effect of ischemia by partial vessel occlusion on neurogenic trigonal and detrusor function in connec-

tion with impaired NO production by accumulated endogenous NOS inhibitors and decreased NOS activity. MATERIALS AND METHODS

This study complied with animal welfare regulations at our institution. Experimental protocol. Japanese White male rabbits weighing approximately 2.5 kg were divided into 2 weight matched groups, including 10 each in a control and an ischemia (IS) group. With the rabbits under sodium pentobarbital anesthesia (25 mg/kg intravenously) the bilateral common iliac arteries were exposed and partially occluded by vessel occluders (Unique Medical Co., Ltd., Tokyo, Japan) (4 mm inner diameter) via an abdominal midline incision. Control rabbits underwent sham surgery without placing occluders. Two weeks later after measuring body weight the animals were again anesthetized by sodium pentobarbital (25 mg/kg intravenously). Bladder blood flow was measured with a laser Doppler probe connected to a TBF-LN1 (Unique Medical Co., Ltd.) laser Doppler flow meter at 3 bladder dome sites and average blood flow was recorded. The flow meter was calibrated against an internal standard reading flow in ml per minute per 100 gm tissue. Systemic blood pressure was monitored via the right carotid artery. Blood was obtained via the central ear artery. The animals were sacrificed with an overdose of sodium pentobarbital, and the lower urinary tract was removed en bloc and maintained in ice-cold modified Krebs solution. In the bladder, which was opened by an anterior longitu-

Accepted for publication April 11, 2003. * Requests for reprints: Department of Urology and Reproductive Medicine, Graduate School, Tokyo Medical and Dental University, 1–5-45, Yushima, Bunkyo-ku, Tokyo, 113-8519, Japan. 1415

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IMPAIRMENT OF TRIGONAL RELAXATION WITH ISCHEMIA

dinal median incision, the detrusor and trigone were separated at the level of the ureteral orifices. Detrusor strips from the anterior dome wall were cut transversely and trigonal strips were obtained in an oblique direction from the internal urethral orifice toward 1 ureteral orifice. For measuring NOS activity endogenous methylarginines and L-arginine part of the tissues was frozen in liquid nitrogen and stored at – 80C. Functional studies. All strips were approximately 5 ⫻10 mm and weighed 60 to 80 mg. Electrical field stimulation (EFS) was performed at supramaximum voltage 0.3 milliseconds in duration at frequencies of 0.5 to 20 Hz for 10 seconds with a 3-minute interval in the presence of 1 ␮M atropine, 10 ␮M guanethidine and 10 ␮M ␣,␤-methylene adenosine triphosphate (ATP) (in the detrusor) to decrease contractions induced by potential excitatory mediators.5, 8 The relaxation response to EFS during contractions with 10 ␮M prostaglandin F2␣ (PGF2␣) in the detrusor and 10 ␮M phenylephrine (PE) in the trigone were recorded. Frequency-response curves to EFS were obtained before and after treatment with 3 mM L-arginine, 100 ␮M L-NOARG (NG-nitro-L-arginine), 1 to 100 ␮M L-NMMA, 1 to 100 ␮M ADMA, 100 ␮M SDMA or 1 ␮M tetrodotoxin (TTX). Subsequently the relaxation response to sodium nitroprusside (SNP) were examined before and after treatment with NOS inhibitors or 10 ␮M ODQ (1H-[1,2,4] oxadiazolo [4,3-a] quinoxalin-1-one) as a soluble guanylate cyclase inhibitor during the contraction with 10 ␮M PE. Relaxation is expressed as a percent of the precontractions with 10 ␮M PGF2␣ in the detrusor and 10 ␮M PE in the trigone. The composition of modified Krebs solution was 118.0 mM NaCl, 4.7 mM KCl, 1.2 mM MgSO4 䡠 7H2O, 2.5 mM CaCl2 䡠 2H2O, 1.2mM KH2PO4, 25.0 mM NaHCO3 and mM 10.0 glucose. Measurement of cyclic guanosine monophosphate (cGMP). Trigonal and detrusor preparations were suspended in organ bath under 1 gm tension. The level of cGMP was measured in unstimulated preparations and in preparations subjected to 10 Hz EFS for 30 seconds under the 10 ␮M PE contraction in the trigone and 10 ␮M PGF2␣ in the detrusor, and determined using radioimmunoassay (Yamasa Shoyu Co., Tokyo, Japan), as previously described.8 All experiments were performed in the presence of 10 ␮M IBMX (3-isobutyl-1methylxantine). Baseline production was considered the value without EFS. Net production is expressed as the difference between EFS production with and without 100 ␮M L-NOARG. The amount of protein was determined using protein assay reagent (BioRad Laboratories, Hercules, California). The cGMP level is expressed in pmol/mg protein. Measurement of NOS activity. NOS activity was measured by determining the conversion of [3H] L-arginine to [3H] L-citrulline, as previously described.5, 8 To determine the effects of authentic L-NMMA, ADMA and SDMA on NOS activity parallel samples were processed in the presence of various concentrations (0.1 to 100 ␮M) of methylarginines and L-NOARG. Enzyme activity is expressed in pmol citrulline per mg protein per minute. Determination of L-arginine and methylarginines. The content of L-arginine, L-NMMA, ADMA and SDMA in the detrusor, trigone and plasma obtained from the 2 groups was determined by automated high performance liquid chromatography, as previously described.2, 9 The tissue contents of L-arginine and methylarginines is shown as nmol/gm wet weight. To estimate the apparent concentration in ␮M in tissues the tissue water content was determined by the difference between the wet and dry weights.5, 8, 9 We used [3H] L-arginine (specific activity 37 MBq). Chemicals were dissolved in distilled water, except IBMX and ODQ, which were dissolved in dimethyl sulfoxide. Deviations from the mean regarding the response curves were statistically analyzed by factorial 2-way ANOVA. The potency of L-NOARG, L-NMMA and ADMA on NOS activity were compared in terms of IC50 values, which were concentrations

producing 50% inhibition of NOS activity. Michaelis constant (Km) and maximum initial velocity (Vmax) values were estimated by nonlinear analysis of the model, V ⫽ (Vmax ⫻ [S])/(Km ⫹ [S]), in which V is initial velocity in pmol citrulline per mg protein per minute and S is the L-arginine concentration in ␮M. The Student 2-tailed t test for unpaired data was used with statistical significance at p ⬍0.05. RESULTS

Table 1 shows baseline data. Mean arterial pressure and body weight in the IS group were not different from those in the control group, while bladder and trigonal weights were significantly greater in the IS group (p ⬍0.05). Bladder blood flow in the IS group was significantly decreased compared with the control group under the empty bladder condition (p ⬍0.01). EFS and SNP induced responses. The mean trigonal contractile response to 10 ␮M PE ⫾ SEM in the control and IS groups was 1.78 ⫾ 0.26 and 1.83 ⫾ 0.29 gm, respectively, which was not significantly different. In the presence of 1 ␮M atropine and 10 ␮M guanethidine EFS caused frequency dependent relaxation of trigonal strips from each group during the 10 ␮M PE induced contraction, which was abolished by 1 ␮M TTX or significantly decreased by 100 ␮M L-NOARG. EFS induced relaxation in the IS group was significantly decreased compared with that in the control group (fig. 1). The maximum relaxation at 10 Hz was 27.7% ⫾ 3.9% in the control and 15.5% ⫾ 3.9% in the IS group in 7 preparations each (p ⬍0.01). Pretreatment with 3 mM. L-arginine for 30 minutes significantly improved but did not normalize impaired relaxation in the IS group at low frequencies (fig. 1). Trigonal relaxation caused by SNP as a NO donor was not different between the 2 groups but it was significantly inhibited by 10 ␮M ODQ (p ⬍0.01, fig. 2). In detrusor strips precontracted with 10 ␮M PGF2␣ in the presence of 1 ␮M atropine, 10 ␮M guanethidine and 10 ␮M␣,␤-methylene ATP all preparations from the 2 groups did not show any relaxation or contraction by EFS that were not modified by 3 mM L-arginine and 100 ␮M L-NOARG in 5 preparations each (data not shown). cGMP production. EFS significantly increased cGMP contents in the trigone (p ⬍0.05) but not in the detrusor in the 2 groups. Baseline and stimulated cGMP production with EFS were significantly decreased in the IS group. Net production was also significantly lower in the IS group (fig. 3). NOS activity. NOS activity was greatly decreased by removing Ca2⫹ and adding 2 mM ethylenediaminetetraacetic acid in the medium, suggesting that NOS activity was mainly Ca2⫹ dependent in the trigone and detrusor in each group (data not shown). Results referred to Ca2⫹ dependent NOS activity. In each region NOS activity was significantly lower in the IS than in the control group (p ⱕ0.01). Moreover, in each group NOS activity in the detrusor was significantly lower than in the trigone (p ⱕ0.05, table 2). In experiments done at L-arginine concentration of 0 to 3,000 ␮M [3H] L-citrulline was generated according to Michaelis-Menten kinetics. Mean apparent Km and Vmax in the trigone were estimated to be 9.3 ⫾ 1.7 ␮M and 28.3 ⫾ 2.9 pmol citrulline per mg protein per minute in 5 preparations in the control TABLE 1. MAP, body weight, wet weight and bladder wall blood flow in control and IS groups MAP (mm Hg) Body wt (kg) Wt (mg): Trigone Bladder Bladder blood flow (ml/min/100 gm)

Mean Control ⫾ SEM

Mean IS ⫾ SEM

91.7 ⫾ 4.8 2.76 ⫾ 0.4

94.1 ⫾ 3.2 2.82 ⫾ 0.3

157 ⫾ 18 2,694 ⫾ 246 14.3 ⫾ 3.8

225 ⫾ 24 3,695 ⫾ 233 6.0 ⫾ 1.9

p Value

⬍0.05 ⬍0.05 ⬍0.01

IMPAIRMENT OF TRIGONAL RELAXATION WITH ISCHEMIA

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FIG. 1. EFS induced trigonal relaxation during contraction with 10 ␮M PE in control and IS groups, and effects of 100 ␮M L-NOARG and 3 mM L-arginine. Data points represent mean measurements ⫾ SEM in 5 to 7 strips in different animals. Vertical bars represent SEM. Double asterisks indicate ANOVA p ⬍0.01 vs corresponding curves in untreated control group. Single pound sign indicates ANOVA p ⬍0.05 vs corresponding curves in untreated IS group. Double pound signs indicate ANOVA p ⬍0.01 vs corresponding curves in untreated IS group.

FIG. 3. Cyclic GMP measured under baseline condition and stimulated production with EFS in control and IS groups in trigone (A) and detrusor (B). Results are shown as mean ⫾ SEM of 5 or 6 determinations in different animals. Vertical bars represent SEM. Single asterisk indicates significantly different (p ⬍0.05). Double asterisks indicate significantly different (p ⬍0.01).

FIG. 2. SNP induced relaxation during contraction with 10 ␮M PE in trigonal strips of control and IS groups, and effect of 10 ␮M ODQ. Data points represent mean measurements ⫾ SEM in 5 to 7 strips in different animals. Vertical bars represent SEM. Double asterisks indicate ANOVA p ⬍0.01 vs corresponding curves in untreated control and IS groups.

group, and 10.4 ⫾ 1.9 ␮M and 18.7 ⫾ 2.8 pmol citrulline per mg protein per minute in 5 preparations in the IS group, respectively. Also, Km and Vmax values in the detrusor were estimated to be 10.3 ⫾ 2.3 ␮M and 19.4 ⫾ 2.9 pmol citrulline per mg protein per minute in 5 preparations in the control group, and 11.0 ⫾ 2.7 ␮M and 11.9 ⫾ 2.3 pmol citrulline per mg protein per minute in 5 preparations in the IS group, respectively. In each region Vmax but not Km was significantly lower in the IS than in the control group (p ⬍0.01).

Furthermore, in each group Vmax but not Km was significantly lower in the detrusor than in the trigone (p ⬍0.05). L-arginine and methylarginines determination. The contents of L-NMMA and ADMA in the trigone and detrusor were approximately 2 and 3-fold higher in the IS than in the control group, respectively (p ⬍0.01, table 3). However, there were no significant differences in L-arginine and SDMA contents between the 2 groups. Tissue water contents in the trigone and detrusor was 0.811 ⫾ 0.008 and 0.807 ⫾ 0.01 ml/gm wet weight in the control group, and 0.815 ⫾ 0.009 and 0.816 ⫾ 0.007 ml/gm wet weight in the IS group, respectively, in 7 preparations each. These values were not significantly different. Table 3 lists the estimated concentration in ␮M of L-arginine, L-NMMA, ADMA and SDMA based on tissue water content. The concentration of L-NMMA and ADMA in the trigone and detrusor were higher in the IS group with no significant changes in L-arginine or SDMA. On the other hand, the plasma concentration of L-arginine and

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IMPAIRMENT OF TRIGONAL RELAXATION WITH ISCHEMIA

TABLE 2. NOS activity, and inhibitory potency of L-NOARG, LNMMA and ADMA in tissues of control and IS groups Group

Mean NOS Activity ⫾ SEM Mean IC50 ⫾ SEM (␮M)† (pmol citrulline/mg L-NOARG L-NMMA ADMA protein/min)*

Trigone: Control IS p Value

21.6 ⫾ 2.8 14.1 ⫾ 2.5 ⬍0.01

0.94 ⫾ 0.07 8.23 ⫾ 1.01 12.1 ⫾ 1.45 1.13 ⫾ 0.11 9.10 ⫾ 1.23 13.1 ⫾ 1.71

Detrusor: Control 14.3 ⫾ 2.5 1.01 ⫾ 0.10 9.25 ⫾ 1.34 13.0 ⫾ 1.22 IS 8.8 ⫾ 1.8 1.10 ⫾ 0.18 10.5 ⫾ 1.15 14.0 ⫾ 1.53 p Value ⬍0.01 Vs control, ⬍0.05 Vs trigone Mean of 6 or 7 determinations in different animals. * Measured in presence of 2 mM reduced nicotinamide adenine dinucleotide phosphate, 30 U/mL calmodulin, 5 ␮M flavin adenine dinucleotide, 14 ␮M tetrahydrobiopterin and 20 ␮M L-arginine. † Determined by linear interpolation.

TABLE 3. Contents and concentrations of L-arginine, L-NMMA, ADMA and SDMA in tissues of control and IS groups Group

L-Arginine

L-NMMA

ADMA

SDMA

FIG. 4. Inhibitory effects of authentic L-NMMA and ADMA on trigonal relaxation induced by EFS in control group. Data points represent mean ⫾ SEM of measurements in 5 to 7 strips in different animals. Vertical bars represent SEM. Single asterisk indicates ANOVA p ⬍0.05 vs corresponding curves in untreated control group. Double asterisks indicate ANOVA p ⬍0.01 vs corresponding curves in untreated control group.

Mean contents ⫾ SEM (nmol/gm wet wt)* Trigone: Control IS

287 ⫾ 32 298 ⫾ 27

1.37 ⫾ 0.11 1.23 ⫾ 0.13 2.41 ⫾ 0.16* 3.44 ⫾ 0.23*

0.43 ⫾ 0.06 0.47 ⫾ 0.05

Detrusor: Control IS

293 ⫾ 24 303 ⫾ 35

1.44 ⫾ 0.08 1.19 ⫾ 0.12 2.34 ⫾ 0.16* 3.47 ⫾ 0.13*

0.44 ⫾ 0.06 0.51 ⫾ 0.11

Mean concentration ⫾ SEM (␮M) Trigone: Control IS

354 ⫾ 39 366 ⫾ 33

1.69 ⫾ 0.14 1.52 ⫾ 0.16 2.96 ⫾ 0.20* 4.22 ⫾ 0.28†

0.53 ⫾ 0.07 0.58 ⫾ 0.06

Detrusor: Control IS

363 ⫾ 30 371 ⫾ 43

1.78 ⫾ 0.10 1.48 ⫾ 0.15 2.87 ⫾ 0.20* 4.25 ⫾ 0.16†

0.55 ⫾ 0.07 0.63 ⫾ 0.13

Plasma: Control 158 ⫾ 19 0.11 ⫾ 0.01 0.64 ⫾ 0.05 0.36 ⫾ 0.04 IS 163 ⫾ 24 0.14 ⫾ 0.02 0.68 ⫾ 0.06 0.30 ⫾ 0.03 Difference between wet weight of preparation and dry weight after complete lyophilization was considered total tissue water content and based on tissue water content, apparent concentrations of tissue L-arginine and methylarginines were calculated. * Mean of 6 or 7 determinations in different animals. † Significantly different vs control (p ⬍0.01).

the 3 methylarginine derivatives remained unchanged following ischemia (table 3). Effects of authentic methylarginines on EFS induced relaxation and NOS activity. EFS induced trigonal relaxation was inhibited by authentic L-NMMA and ADMA (10 and 100 ␮M) but not by SDMA even at the high concentration of 100 ␮M (data not shown) in 6 preparations each (fig. 4). Inhibition with 100 ␮M L-NMMA or 100 ␮M ADMA was undetectable in the presence of 3 mM L-arginine but not 3 mM D-arginine. Three authentic methylarginines at 100 ␮M each failed to modify SNP induced relaxation in the 2 groups (data not shown). NOS prepared from the trigone and detrusor in each group was inhibited by authentic L-NMMA and ADMA in a concentration dependent manner but not by SDMA even at the high concentration of 100 ␮M (fig. 5). The inhibitory potency of L-NMMA and ADMA was compared with that of L-NOARG in terms of IC50 in ␮M (table 2). The rank order of potency in each region of the 2 groups was L-NOARG ⬎ L-NMMA ⬎ ADMA (L-NOARG vs L-NMMA and L-NOARG vs ADMA p ⬍0.01, and L-NMMA vs ADMA p ⬍0.05). IC50 values in the IS group were not significantly different from corresponding control values.

FIG. 5. Effects of L-NOARG, L-NMMA, ADMA and SDMA on NOS activity in control (right) and IS (left) groups in trigone (A) and detrusor (B). Values are expressed as percent of control NOS activity. Data points represent mean ⫾ SEM of 6 or 7 measurements in different animals. Vertical bars represent SEM. Single asterisk indicates p ⬍0.05 vs untreated control NOS activity. Double asterisks indicate p ⬍0.01 vs untreated control NOS activity. Single pound sign indicates p ⬍0.05 vs untreated IS NOS activity. Double pound signs indicate p ⬍0.01 vs untreated IS NOS activity.

DISCUSSION

The inhibition of EFS induced trigonal relaxation with TTX and L-NOARG in each group suggests that relaxation is characterized as neurogenic and NO dependent. Moreover,

IMPAIRMENT OF TRIGONAL RELAXATION WITH ISCHEMIA

trigonal relaxation caused by EFS but not by SNP was impaired in the IS group, assuming that ischemia results in impaired neurogenic NO production. This assumption is partially supported by the finding that baseline and stimulated cGMP production with EFS was significantly decreased in the IS group. It is well established that cGMP generation is widely used as an index of NO biosynthesis.10 Exogenous L-arginine supplementation partially restored impaired trigonal relaxation in the IS group. Endogenous L-arginine concentration in the trigone remained unchanged following ischemia and it was in the 300 to 400 ␮M range in each group. Furthermore, the determined Km for NOS activity was approximately 10 ␮M in the IS group. That is, although the apparent L-arginine concentration was enough to saturate NOS in the IS group, NO mediated trigonal relaxation was significantly enhanced by exogenous L-arginine, which is in line with the previously reported finding.8 The accumulation of endogenous NOS inhibitors may partly explain the findings, termed the arginine paradox.11 The apparent concentration of L-NMMA plus ADMA in the trigone was increased from 3.2 ␮M in the control group to 7.2 ␮M in the IS group. However, in the enzymatic study 7.2 ␮M methylarginines did not significantly inhibit NOS activity in the presence of more than 300 ␮M L-arginine (data not shown). Rather, it significantly inhibited NOS by approximately 41% in the presence of 20 ␮M L-arginine. If endogenous L-arginine in the vicinity of NOS is more than 300 ␮M, it would overcome any competitive inhibition of NOS by L-NMMA and ADMA. On the other hand, in the functional study adding authentic 1.3 ␮M L-NMMA plus 2.7 ␮M ADMA, obtained by calculating the difference of methylarginines between the 2 groups, significantly inhibited NO mediated trigonal relaxation (data not shown). These findings suggest that endogenous L-arginine may be compartmentalized in cells and poorly accessible to NOS, and the accumulation of NOS inhibitors in the vicinity of NOS may modulate NO mediated lower urinary tract function. Moreover, because methylarginines are concentrated within cells,12 intracellular concentrations would be possibly higher than estimated concentrations. Recently it was reported that the ADMA level in cultured endothelial cells is approximately 10-fold higher than in medium.13 The L-arginine-to-methylarginine ratio in the vicinity of NOS may be different from the total L-arginine-to-methylarginine ratio in tissues. The other possibility is that, if extracellular L-arginine transported into the cell mediated via system y⫹ transporter is preferentially delivered to NOS, pretreatment with a high concentration of L-arginine may act by competing with methylarginines to increase NO production. A more recent report describes that caveolae also contain the y⫹ arginine transporter and suggests that L-arginine may be delivered directly from the extracellular pool to endothelial NOS (eNOS) via the y⫹ transporter.14 Further studies of the intracellular localization and regulation of transporter, methylarginines and NOS are required to clarify NOS regulation by methylarginines. In contrast to the trigone, attempts to find neurogenic and NO mediated relaxation have failed irrespective of the existence of NOS enzyme and EFS did not increase cGMP contents in the detrusor of either group. These findings may have resulted from a low level of soluble guanylate cyclase activity in the detrusor muscle.15 Recently oxyhemoglobin, a NO scavenger and ODQ, an inhibitor of soluble guanylate cyclase, induced bladder overactivity,16 suggesting that NO/ cGMP may be involved in regulation of the threshold for afferent firing in the bladder. Also, NO appears to have a central role in the regulation of bladder dome and base blood flow.17 Bladder ischemia has been shown to produce low compliance and hyperreflexia.18 Therefore, accumulated endogenous NOS inhibitors and decreased NO production under the ischemic state are possibly related to bladder overactivity. To clarify this hypothesis the effects of

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methylarginines and L-arginine on cystometric parameters, such as the micturition interval, bladder compliance and contraction, should be investigated. Interestingly the ratio and concentration of methylarginines differed in plasma and tissues. In plasma ADMA and SDMA were the major circulating form and L-NMMA was considerably lower, whereas L-NMMA concentration was approximately equal to that of ADMA in control tissues. Also, in this study plasma L-NMMA and ADMA did not change following ischemia, suggesting that the plasma methylarginine level does not necessarily reflect methylarginine in cells and tissues. However, many other researchers have reported elevated plasma ADMA in multiple disorders in which NOS dysfunction has been implicated, while little attention has been given to L-NMMA. Moreover, the potency of L-NMMA on NOS was greater than that of ADMA. Therefore, L-NMMA should be investigated in the regulation of NOS in cells and tissues. In this study the Vmax of NOS activity in the trigone and detrusor was significantly lower in the IS group. Furthermore, methylarginines and L-arginine were undetectable in the partially purified NOS preparation (unpublished data). These findings lead us to assume that the decreased NOS activity following ischemia would be a reflection of decreased NOS protein. This speculation may be supported by the demonstration that pulmonary hypertension induced after chronic ischemia is associated with decreased eNOS activity resulting from reduced eNOS protein and mRNA expressions.19 In conclusion, this study suggests that impaired neurogenic NO mediated trigonal relaxation following ischemia is closely related to decreased NOS activity, and the increased accumulation of L-NMMA and ADMA. L-phenylephrine hydrochloride, guanethidine sulfate, atropine sulfate, ␣,␤-methylene ATP, L-citrulline, L-arginine hydrochloride, L-NMMA, ADMA, SDMA, PGF2␣, HEPES, sucrose, ethylenediaminetetraacetic acid, dithiothreitol, leupeptin, reduced nicotinamide adenine dinucleotide phosphate, CaCl2, IBMX, ODQ, calmodulin, MgCl2 and bovine serum albumin were obtained from Sigma Chemical Co., St. Louis, Missouri. L-NOARG was obtained from Research Biochemicals, Inc., Natik, Massachusetts. SNP was obtained from Wako Pure Chemicals, Tokyo, Japan. TTX was obtained from Sankyo Co., Tokyo, Japan. [3H] L-arginine was obtained from Amersham Pharmacia Biotech, Little Chalfont, United Kingdom. REFERENCES

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