ACCUMULATED ENDOGENOUS NITRIC OXIDE SYNTHASE INHIBITORS IN INHIBITING URETHRAL RELAXATION FOLLOWING ESTROGEN SUPPLEMENTATION IN OVARIECTOMIZED RABBITS

0022-5347/04/1721-0360/0 THE JOURNAL OF UROLOGY® Copyright © 2004 by AMERICAN UROLOGICAL ASSOCIATION

Vol. 172, 360 –364, July 2004 Printed in U.S.A.

DOI: 10.1097/01.ju.0000121692.52724.c5

ACCUMULATED ENDOGENOUS NITRIC OXIDE SYNTHASE INHIBITORS IN INHIBITING URETHRAL RELAXATION FOLLOWING ESTROGEN SUPPLEMENTATION IN OVARIECTOMIZED RABBITS TETSUO OKUNO, HITOSHI MASUDA, TOSHIHIKO TSUJII, KAZUNORI KIHARA, YUKINAO YAMAUCHI AND HIROSHI AZUMA* From the Department of Urology and Reproductive Medicine and Department of Biosystem Regulation, Institute of Biomaterials and Bioengineering, Graduate School, Tokyo Medical and Dental University, Tokyo, Japan

ABSTRACT

Purpose: We investigated the possible role of the endogenous nitric oxide (NO) synthase (NOS) inhibitors NG-monomethyl-L-arginine (L-NMMA) and asymmetrical NG, NG-dimethyl-Larginine (ADMA) in inhibiting urethral relaxation following estrogen supplementation in ovariectomized rabbits. Materials and Methods: A total of 16 mature Japanese White female rabbits were divided into 2 groups. In the control group rabbits were sacrificed 2 weeks after bilateral ovariectomy. In the estrogen group estradiol was administered subcutaneously for 2 weeks with the aid of sustained release pellet from 2 weeks after ovariectomy until sacrifice. Isolated urethra was cut into transverse strips for functional study and processed to determine endogenous NOS inhibitors, NOS activity, dimethylarginine dimethylaminohydrolase (DDAH) activity as a metabolizing enzyme of endogenous NOS inhibitors and cyclic guanosine monophosphate production. Results: Electrical field stimulation produced NO mediated and neurogenic relaxation of the urethral strip in the presence of guanethidine and atropine under contraction with phenylephrine. Relaxation was significantly decreased in the estrogen group and accompanied by decreased cyclic guanosine monophosphate production. Sodium nitroprusside induced relaxation was not different between the 2 groups. The content of L-NMMA plus ADMA in the urethra was significantly increased in the estrogen group. Ca2⫹ dependent NOS activity in the urethra remained unaffected. DDAH activity was significantly lower in the estrogen group. Conclusions: Estrogen supplementation leads to decreased NO mediated and neurogenic urethral relaxation through the accumulation of L-NMMA and ADMA in the urethra. The accumulation of NOS inhibitors is possibly brought about by impaired DDAH activity. KEY WORDS: urethra, estradiol, omega-N-methylarginine, N,N-dimethylarginine, dimethylargininase

Estrogen has been used clinically for the treatment of urinary incontinence in postmenopausal women.1, 2 Many groups have reported the effects of estrogen on functional and structural changes in the urethra. In female rabbits estrogen increases urethral tissue mass and the sensitivity of urethral smooth muscle to autonomic drugs.3 In addition, it has been reported that estrogen treatment increases ␣2adrenoceptors in the urethra and increases contractility in the female rabbit urethra.4 Recently nitric oxide (NO) has been identified as an important mediator in urethral relaxation.5, 6 Several investigators reported the effects of estrogen on the L-arginine/NO system and on NO mediated relaxation.7, 8 Takahashi et al noted that estrogen treatment might cause a decrease in NO synthase (NOS) activity, contributing to the decrease in urethral relaxation in the rabbit.7 However, the mechanism of decreased urethral relaxation and inhibited L-arginine/NO pathway by estrogen has not been explained clearly. NG-monomethyl-L-arginine (L-NMMA) and asymmetrical G N , NG-dimethyl-L-arginine (ADMA) are endogenously produced methylarginines that have a role as NOS inhibitors. These methylarginines compete with L-arginine for NOS and may decrease NO production.9 In recent years many studies

revealed that the accumulation of these inhibitors might contribute to some pathological state in humans and experimental animals.10⫺12 Previously we have observed that impaired neurogenic relaxation with ischemia was due to the accumulation of endogenous NOS inhibitors and decreased NOS activity in the rabbit proximal urethra.6 However, to our knowledge there is no information on the role of endogenous NOS inhibitors in the urethra under estrogen supplementation. Dimethylarginine dimethylaminohydrolase (DDAH) is an enzyme that hydrolyzes L-NMMA and ADMA.13 Thus, it may provide a mechanism for controlling NO production by regulating the levels of these inhibitors. In the current study we evaluated the effect of estrogen on NO mediated urethral relaxation in connection with changes in endogenous NOS inhibitors and DDAH activity in tissue. MATERIALS AND METHODS

Mature Japanese White female rabbits weighing 2.5 to 3.0 kg were divided into 2 groups, that is a control group of 8 and an estrogen group of 8. Bilateral ovariectomy through an abdominal midline incision was performed in all animals under anesthesia with intravenous injection of sodium pentobarbitone (25 mg/kg). In the control group rabbits were sacrificed 2 weeks after ovariectomy. In the estrogen group we embedded subcutaneously a sustained release estradiol pellet for 2 weeks from 2 weeks after ovariectomy until sac-

Accepted for publication January 2, 2004. * Correspondence: Department of Biosystem Regulation, Institute of Biomaterials and Bioengineering, Graduate School, Tokyo Medical and Dental University, 2–3-10 Kanda-Surugadai, Chiyoda-Ku, Tokyo, 101-0062, Japan. 360

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rifice. At the time of the experiment peripheral blood was collected for the hormone assay and animals were sacrificed with an overdose of sodium pentobarbitone. The entire lower urinary tract was removed and maintained in a Petri dish containing ice-cold modified Krebs solution. For the functional study the urethra was dissected free of adherent tissues and cut into transverse strips approximately 2 mm wide ⫻ 6 mm long and weighing about 40 mg. The strips were suspended vertically between 2 parallel platinum electrodes in 10 ml organ chambers filled with modified Krebs solution maintained at a mean temperature ⫾ SEM of 37C ⫾ 0.5C and continuously bubbled with 95% O2 and 5% CO2. An end of each strip was tied to a force displacement transducer (TB-612T, Nihon Kohden Kogyo Co., Ltd., Tokyo, Japan) to record changes in isometric tension on a pen writing oscillograph (R64, Rika Denki Kogyo Co., Tokyo, Japan) according to a previously described method.6 Relaxations in response to electrical field stimulation (EFS) and sodium nitroprusside (SNP) during the contraction caused by phenylephrine (PE) (5 ␮M) were observed. EFS was produced by an electronic stimulator (SEN-3201, Nihon Kohden Kogyo Co., Ltd.), which delivered trains of rectangular pulses (supramaximum voltage 0.3 milliseconds in duration at frequencies of 0.5 to 20 Hz for 10 seconds). EFS was applied every 3 minutes in the presence of guanethidine (10 ␮M) and atropine (1 ␮M) to eliminate adrenergic and cholinergic components. Following a 60-minute washout period the strips were again contracted with PE (5 ␮M) and responses to SNP were examined. To characterize the responses EFS was applied to the strips after treatment with L-arginine (3 mM), L-NMMA (1 to 100 ␮M), ADMA (1 to 100 ␮M), NG-nitro-L-arginine (LNA) (100 ␮M) or tetrodotoxin (TTX) (1 ␮M). Relaxation induced by EFS and SNP is expressed as a percent of the PE contraction. The contents of L-arginine, L-NMMA, ADMA and SDMA (symmetrical NG, N⬘G-dimethyl-L-arginine) in the urethra were determined by automated high performance liquid chromatography according to a previously described method.10, 14 The contents of L-arginine and methylarginines are shown as nmoles and pmoles/gm wet weight, respectively. To estimate the apparent concentrations of L-arginine and methylarginines in the urethra tissue water content was determined according to a previously described method.14 Cyclic guanosine monophosphate (GMP) levels were measured in unstimulated preparations and in preparations subjected to 10 Hz EFS for 30 seconds under the PE (5 ␮M) contraction in the presence of 10 ␮M 3-isobutyl-l-methylxanthine (IBMX). The cyclic GMP level was determined by a previously described method.6 NOS activity in the urethra was measured by determining the conversion of [3H]-L-arginine to [3H]-L-citrulline.15 After thawing tissue was homogenized in a Polytron (Kinematica, Luzern, Switzerland) at maximal speed for 15 seconds each ⫻ 4 to a 25% homogenate in the buffer consisting of 50 mM tris-HCl (pH 7.4), 10 mM cyclohexylaminopropanesulfonic acid, 2 mM ethylenediaminetetraacetic acid (EDTA), 1 mM DL-dithiothreitol, 1 mM phenylmethyl sulfonyl fluoride, 0.001 mM pepstatin A and 0.001 mM leupeptin. The homogenate was centrifuged at 10,000 ⫻ gravity for 20 minutes at 4C and the supernatant was decanted from the pellet. The reaction mixture was incubated at 37C for 60 minutes in a shaking water bath. Samples were applied to a 1 ml column of a Dowex AG50W-X8 (Na⫹ form) to remove unmetabolized [3H]-L-arginine. The columns were then washed with 1.5 ml water. [3H]-L-citrulline was quantified in the flow through fractions using a liquid scintillation counter (Tri-Carb 2750TR/LL, Packard Instrument Co., Meriden, Connecticut). DDAH activity was measured by monitoring the formation of [3H]-L-citrulline from L-[3H]-NMMA.13 After thawing tissue was homogenized in medium consisting of 0.1 M Na-phosphate buffer (pH 6.5), 1 mM phenylmethyl sulfo-

nyl fluoride, 1 ␮M pepstatin A and 2 ␮M leupeptin. The homogenate was then centrifuged and supernatant was collected. The reaction mixtures consisted of 90 ␮l supernatant, 0.01 ␮Ci[3H]-L-NMMA, 0.1 ␮M L-NMMA and 20 mM EDTA. DDAH activity was assayed in the same manner as the determination of NOS activity after the mixture was incubated at 37C for 120 minutes. Protein and DNA concentrations were determined according to a previously described method.6, 14 Plasma estradiol concentration was measured using an estradiol radioimmunoassay kit (DPC Co., Tokyo, Japan). The chemicals used were PE, guanethidine sulfate, L-citrulline, L-arginine hydrochloride, L-NMMA, ADMA, EDTA, DL-dithiothreitol, leupeptin, reduced nicotinamide adenine dinucleotide phosphate (NADPH), calmodulin, IBMX and Dowex AG50W-X8 (Na⫹ form), SNP, TTX, estradiol pellets (17␤-estradiol, 10 mg per pellet for 90-day release), [3H]-L-citrulline and [3H]-LNMMA [NG-monomethyl-L-arginine (2,3,4-3H)] (specific activity 2.00 TBq/mmol). The radiochemical purity of [3H]-LNMMA, which was determined elsewhere by high performance liquid chromatography, was 97.6%. Results are shown as the mean ⫾ SEM. Frequency-response curves were statistically analyzed by 2-way ANOVA. Student’s t test for unpaired data was used and statistical significance was determined at p ⬍0.05. Potencies of L-NMMA and ADMA on NOS activity were compared in terms of IC50 values, that is concentrations producing 50% inhibition of NOS activity. RESULTS

Baseline data. The table lists body weight, and wet weight of the bladder, urethra and uterus of the rabbits at sacrifice. Body weight was not significantly different but wet weight of the bladder, urethra and uterus was significantly greater in the estrogen group than in controls (p ⬍0.01). Plasma estradiol in the estrogen group was 84.7 ⫾ 1.5 pg/ml, whereas it was less than 10 pg/ml in the control group (5 preparations each). Tissue water contents were 0.811 ⫾ 0.003 ml/gm wet weight in the control group and 0.819 ⫾ 0.003 ml/gm wet weight in the estrogen group (5 preparations each). DNA contents in the urethra were 1,030 ⫾ 210 ␮g/gm wet weight in controls and 870 ⫾ 100 ␮g/gm wet weight in the estrogen group with no difference between the 2 groups. Mechanical responses of the urethra. Contractile responses induced by 5 ␮M PE in the control and estrogen groups were 1.68 ⫾ 0.20 g and 1.74 ⫾ 0.22 gm, respectively. These values were not significantly different. EFS at frequencies of 0.5 to 20 Hz produced transient relaxation of the urethral strips in each groups, which had been pre-contracted with 5 ␮M PE (fig. 1). Transient relaxations were abolished by pretreatment with 1 ␮M TTX or 100 ␮M LNA. Inhibition of the relaxation with LNA was overcome by pretreatment with 3 mM L-arginine. EFS induced relaxations in the estrogen group were significantly decreased compared with those in the control group. Maximal relaxation was 68.3% ⫾ 2.0% at 20 Hz in the control group and 38.8% ⫾ 4.9% at 20 Hz in the estrogen group (6 preparations each) with significant difference between the 2 groups (p ⬍0.01). Pretreatment with 3

Body weight, plasma estradiol, and wet weight of urethra, bladder and uterus in control and estrogen groups at sacrifice Mean Control ⫾ SEM Body wt (kg) 2.92 ⫾ 0.15 Plasma estradiol (pg/ml) Less than 10 Wet wt (mg): Urethra 283 ⫾ 69 Bladder 2,821 ⫾ 553 Urethra 1,282 ⫾ 384 Five to 8 determinations in different animals. * Significantly different vs control (p ⬍ 0.01).

Mean Estrogen ⫾ SEM 2.93 ⫾ 0.21 84.7 ⫾ 1.5 499 ⫾ 76* 4,434 ⫾ 880* 8,694 ⫾ 577*

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FIG. 1. Representative tracings show relaxation responses to EFS delivered to strips pre-contracted with PE (5 ␮M) in presence of atropine (1 ␮M) and guanethidine (10 ␮M). Relaxation responses were greater in control than in estrogen group. (a), control. (b), estrogen.

mM L-arginine significantly increased relaxation in each group (fig. 2, a). On the other hand, relaxation produced by SNP (0.1 to 100 ␮M) was not different between the 2 groups (fig. 2, b). Exogenously applied ADMA inhibited EFS induced relaxation in a concentration dependent manner without affecting resting tension or the contractile response induced by 5 ␮M PE (fig. 3). Cyclic GMP production. Cyclic GMP contents in the urethra were increased by EFS (10 Hz for 30 seconds) in the 2 groups. Pretreatment with LNA markedly decreased cyclic GMP production. The baseline level, level after EFS and net production of cyclic GMP were significantly lower in the estrogen group (fig. 4). L-arginine and methylarginine contents in the urethra. The values of L-arginine and methylarginines are expressed as pmol/gm wet weight (fig. 5). The content of L-NMMA plus ADMA per gm wet weight in the estrogen group was approximately 2-fold higher than in the control group (p ⬍0.01), whereas L-arginine and SDMA were not significantly different between the 2 groups. The apparent concentrations of L-arginine, L-NMMA and ADMA, which were determined based on tissue water content, were 254 ⫾ 19, 0.6 ⫾ 0.1 and

FIG. 3. Inhibitory effects of ADMA on urethral relaxations induced by EFS in control group. Strips were pretreated with 1 to 100 ␮M ADMA for at least 30 minutes. Results are expressed as percent of PE contraction. Data points represent mean ⫾ SEM of measurements in 5 to 8 strips from 5 to 8 animals. Asterisks indicate ANOVA p ⬍0.01 vs untreated control curve.

FIG. 4. Cyclic GMP measured under baseline condition and EFS in control and estrogen groups. Net cyclic GMP production is expressed as difference between production with EFS and with EFS in presence of 100 ␮M LNA as NOS inhibitor. Baseline cyclic GMP was considered value without EFS. All experiments were performed in presence of 10 ␮M IBMX as nonselective phosphodiesterase inhibitor. Results are shown as mean ⫾ SEM of 5 animals per group. Asterisks indicate significantly different vs control at p ⬍0.01.

FIG. 2. (a), EFS induced relaxation during contraction produced by PE (5 ␮M) in rabbit urethral strips before and after treatment with 3 mM L-arginine in control and estrogen groups. Results are expressed as percent of PE contraction. Data points represent mean ⫾ SEM of measurements in 5 strips from 5 animals. a and b, vs curve for nontreated strips in same group ANOVA p ⬍0.01. Asterisks indicate ANOVA p ⬍0.01 vs control group. (b), SNP induced relaxation during contraction produced by PE (5 ␮M). SNP was given cumulatively after strip achieved steady state tension. Data points represent mean ⫾ SEM of measurements in 6 strips from 6 animals.

0.6 ⫾ 0.1 ␮M in the control group, and 268 ⫾ 10, 0.9 ⫾ 0.1 and 1.2 ⫾ 0.1 ␮M in the estrogen group, respectively. NOS and DDAH activities in the urethra.There were no differences in NOS activities under each condition in the 2 groups (fig. 6). The addition of EDTA or LNA significantly inhibited NOS activity, whereas aminoguanidine as an inducible NOS inhibitor16 failed to modify NOS activity. Exogenously applied L-NMMA and ADMA inhibited NOS activity in a concentration dependent manner (fig. 7). IC50 values were 5.05 ␮M for L-NMMA and 6.42 ␮M for ADMA. DDAH activity in urethras in the estrogen group was significantly decreased, as determined per ␮g DNA (p ⬍0.05) and per mg protein (p ⬍0.01, fig. 8).

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363

FIG. 5. Tissue contents in urethras in control and estrogen groups. (a), L-NMMA, ADMA and SDMA. (b), L-arginine. Results are shown as mean ⫾ SEM of 6 to 8 determinations in 8 animals. Asterisks indicate significantly different (p ⬍0.01).

FIG. 7. Inhibitory effect of L-NMMA and ADMA on NOS activity in 5 control urethras. Values are expressed as percent of control NOS activity. Data points represent mean ⫾ SEM of 5 measurements in 5 animals.

FIG. 6. NOS activity in urethral tissue in control and estrogen groups in 4 preparations each. Activity was also measured in presence of 20 mM EDTA, 100 ␮M NG-nitro-L-arginine (LNA) and 30 ␮M aminoguanidine (AG). All reaction mixtures contained 12 ␮M L-arginine, 1 mM. NADPH, 4 ␮M flavin adenine dinucleotide, 4 ␮M flavin mononucleotide, 10 ␮M tetrahydrobioptelin and 1 mg/l calmodulin. Values are expressed in citrulline production per minute. DISCUSSION

EFS induced urethral relaxations in the control and estrogen groups were observed in the presence of guanethidine and atropine, and they were abolished by pretreatment with TTX or LNA. Inhibition with LNA was overcome by excess L-arginine. These results coincided well with those in the previous report7 and strongly suggest that relaxations are characterized to be neurogenic, nonadrenergic, noncholinergic and NO dependent. The current study revealed that NO mediated and neurogenic urethral relaxation was significantly attenuated in the estrogen group, while relaxation in response to SNP as a NO donor remained unaffected even in the estrogen group. SNP activates soluble guanylate cyclase by releasing NO from its structure and increasing tissue cyclic GMP, thereby, relaxing smooth muscles independent of NOS. These results suggest that estrogen treatment possibly decreases NO production and/or release in the urethra since cyclic GMP production in response to EFS was significantly inhibited in the estrogen group and by LNA as a NOS inhibitor, and the estrogen treatment does not affect NO action downstream the activation of soluble guanylate cyclase. This speculation seems to be partly supported by the previous report.7

FIG. 8. DDAH activity in urethral tissue. (a), expressed per ␮g DNA. Single asterisk indicates significantly different vs control (p ⬍0.05). (b), expressed per mg protein. Double asterisks indicate significantly different vs control (p ⬍0.01). Results are shown as mean ⫾ SEM of 4 determinations in 4 animals.

Recently greater attention has been paid to the role of accumulated endogenous NOS inhibitors such as L-NMMA and ADMA as the cause of various disease states. There have been reports describing the possible involvement of endogenous NOS inhibitors in the intimal hyperplasia,10 peripheral arterial occlusive disease11 and diabetes mellitus.12 In the lower urinary tract we have previously reported that impaired neurogenic relaxation of the rabbit proximal urethra with ischemia was possibly due to the accumulation of endogenous NOS inhibitors and decreased NOS activity.6 In the current study the values of L-NMMA plus ADMA in urethras in the estrogen group were about 2-fold higher than in the control group (p ⬍0.01). Apparent concentrations in the urethra increased from 0.6 to 0.9 ␮M for L-NMMA and from 0.6 to 1.2 ␮M for ADMA after estradiol treatment. Authentic L-NMMA and ADMA at a concentration of 1 ␮M each inhibited NOS activity by 10.0% ⫾ 12.5% and 10.6% ⫾ 7.6%, respectively. Furthermore, the potency to inhibit the NO mediated urethral relaxation in response to EFS was estimated to be 5.4% ⫾ 10.5% to 17.1% ⫾ 8.3% (5 to 8 preparations) at the same concentration of 1 ␮M ADMA, although the magnitude of inhibition was different at different frequencies. We observed a similar result for L-NMMA in the previous report.6 Since methylarginines are considered to be concentrated in the vicinity of NOS6 and the apparent

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concentrations of inhibitors were calculated based on tissue water content, it is possible to assume that increased L-NMMA and ADMA in the urethra following estradiol supplementation inhibit NOS activity and, thereby, attenuate NO mediated relaxation in the urethra. Estrogen supplementation did not change NOS activity. In the presence of EDTA NOS activity decreased to a sixth but aminoguanidine as an inducible NOS inhibitor16 failed to modify the activity. This means that NO is mainly produced through the action of constitutive NOS in the female rabbit urethra. Al-Hijji and Batra reported that cytosolic NOS activity in the rabbit urethra treated with estrogen was decreased compared with ovariectomized rabbits without estrogen treatment.8 The reason for the discrepancy from our data may be due to the differences in the methods of estrogen supplementation and/or determinations of NOS activity. They administered polyestradiol phosphate by a single intramuscular injection, whereas we embedded the sustained release estradiol pellet under the skin. The enzyme preparations were also different. They obtained cytosolic and particulate fraction as the enzyme source and particulate NOS activity remained unaffected even after estrogen treatment.8 On the other hand, we determined the crude enzyme fraction by adjusting the centrifugation of the homogenate. Further investigations are needed to clarify the changes in this enzyme activity. DDAH is an enzyme that hydrolyzes L-NMMA to L-citrulline and monomethylamine, and hydrolyzes ADMA to L-citrulline and dimethylamine.13 This enzyme is widely distributed in tissues17 and it may provide a mechanism for controlling NO synthesis in physiological or disease states by regulating the levels of L-NMMA and ADMA. There have been reports demonstrating that an impaired NO pathway was associated with decreased DDAH activity in hypercholesterolemia and diabetes mellitus.18 In the current study estrogen treatment decreased DDAH activity in urethral tissue to approximately 40%. This decrease in DDAH activity may be the cause of the increase in L-NMMA and ADMA. This seems to be partly supported by the finding that SDMA, which is not a substrate for DDAH, remained unchanged in the current study. Another possible mechanism for the accumulation of methylarginines is activation of the cationic amino acid transport system known as system y⫹.19 L-NMMA, ADMA and SDMA enter cells through the transporter.19 However, it seems unlikely that estrogen treatment affects transporter activity in the female rabbit urethra because the SDMA concentration remained unchanged in the current study. Estrogen replacement has been found to improve urinary incontinence in postmenopausal women.1, 2 The beneficial effects of estrogen on the urethra are based on functional changes as well as structural alterations in the urethra.20 The current study revealed that endogenous NOS inhibitors accumulated in the urethra following estrogen supplementation and, thereby, possibly decreased NO mediated urethral relaxation. This mechanism may have a role in the improvement of urinary incontinence by estrogen supplementation. CONCLUSIONS

Estrogen treatment decreased NO mediated and neurogenic urethral relaxation through an accumulation of endogenous NOS inhibitors in tissue, which would be brought about by decreased DDAH activity as a metabolizing enzyme of these inhibitors. This mechanism may also be beneficial as a therapeutic strategy in stress urinary incontinence in postmenopausal women. PE, guanethidine sulfate, L-citrulline, L-arginine hydrochloride, L-NMMA, ADMA, EDTA, DL-dithiothreitol, leupeptin, NADPH, calmodulin, IBMX and Dowex AG50W-X8 (Na⫹ form) were obtained from Sigma Chemical Co., St. Louis, Missouri. SNP was obtained from Wako Pure Chemicals, Tokyo, Japan. TTX was obtained from Sankyo Co., Tokyo, Japan. Estradiol

pellets were obtained from I. R. A., Sarasota, Florida. [3H]-Lcitrulline was obtained from Amersham Pharmacia Biotech, Little Chalfont, United Kingdom. [3H]-L-NMMA was obtained from Daiichi Pure Chemicals Co., Ibaraki, Japan. [3H]-LNMMA radiochemical purity was determined at Dupon/NEN Boston Analytical Service, Boston, Massachusetts.

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