International Journal of Impotence Research (2003) 15, 355–361 & 2003 Nature Publishing Group All rights reserved 0955-9930/03 $25.00 Role of the -cyclic GMP pathway in regulation of vaginal blood flow

SW Kim1, S-J Jeong2, R Munarriz2,NNKim2, I Goldstein2 and AM Traish2,3*

1Department of Urology, Seoul National University College of Medicine, Seoul, Korea; 2Department of Urology, Boston University School of Medicine, Boston, Massachusetts, USA; and 3Department of Biochemistry, Boston University School of Medicine, Boston, Massachusetts, USA

The regulatory role of nitric oxide (NO) in vaginal perfusion remains unclear. We used specific inhibitors of in the NO-cyclic GMP (NO-cGMP) pathway and investigated their effects on vaginal blood flow in the rabbit. NO synthase (NOS) activity was similar in both the proximal and distal rabbit vagina; whereas, arginase activity was 3.4-fold higher in the distal vagina. Intravenous administration of the NOS inhibitor L-NAME resulted in a 66% reduction in genital tissue oxyhemoglobin and a 53% reduction in vaginal blood flow. This attenuation occurred despite a 20–30% increase in systemic arterial pressure. The arginase inhibitor ABH caused a 2.1-fold increase in genital tissue oxyhemoglobin and 34% increase in vaginal blood flow. The inhibitor 1H-[1,2,4]oxadiazolo[4,3,-a]quinoxalin-1-one and the phosphodiesterase type 5 inhibitor caused in a 37% reduction and a 44% increase in vaginal blood flow, respectively. These observations suggest that the NO-cGMP pathway is an important regulator of vaginal hemodynamics. International Journal of Impotence Research (2003) 15, 355–361. doi:10.1038/sj.ijir.3901038

Keywords: nitric oxide; L-NAME; guanylate cyclase; arginase; female genital arousal; oximetry; laser Doppler flowmetry

Introduction nergic (NANC) relaxation responses that are mediated by nitric oxide (NO) in rabbit clitoral corpus cavernosum. While the NO-cyclic GMP (NO- The arousal phase of the female sexual response cGMP) pathway has been reported to be the major involves, in part, local regulatory mechanisms that mediator of neurogenic relaxation in rat vagina,10 its modulate tone in the clitoral and labial erectile significance in the rabbit has been questioned.11 tissue and the vascular and nonvascular smooth However, these prior studies only investigated 1,2 muscle of the vagina. Immunohistochemical stu- nonvascular smooth muscle relaxation in organ dies in human vaginal tissues have identified nerve baths and did not examine the reactivity of blood fibers containing neuropeptide Y, vasoactive intest- vessels within the vagina. To fully appreciate the inal polypeptide (VIP), (NOS), role of the NO-cGMP pathway in the vagina, the 3 calcitonin gene-related peptide and substance P. regulation of vascular smooth muscle tone must also Previous studies have suggested that VIP may be be studied. involved in the regulation of clitoral and vaginal Thus, the goal of this study was to investigate the smooth muscle tone, but no conclusive experimen- role of NO in regulating vaginal blood flow in vivo. tal evidence of its functional involvement has been To this end, experiments were performed in 3–8 forthcoming. anesthetized female rabbits using inhibitors of 9 Cellek and Moncada demonstrated that electrical NOS, arginase, guanylate cyclase and phosphodies- field stimulation causes nonadrenergic, noncholi- terase type 5 (PDE 5); enzymes that modulate the NO-cGMP pathway. We also measured the enzy- matic activities of NOS and arginase in the vagina. Arginase catalyzes the conversion of L- to *Correspondence: AM Traish, PhD, Laboratory for Sexual and , while NOS utilizes L-arginine as Medicine Research, Department of Urology, Boston Uni- versity School of Medicine, 700 Albany St. W607, Boston, a cosubstrate in the production of NO. Studies in MA 02118, USA other tissues have demonstrated the ability of E-mail: [email protected] arginase to decrease arginine availability for NOS, Received 2 December 2002; revised 2 April 2003; accepted thereby decreasing NO production and smooth 7 April 2003 muscle relaxation.12,13 NO-cGMP in regulation of vaginal blood flow SW Kim et al 356 Methods activity in cytosolic extracts of vaginal tissue was assessed by the method of Ru¨ egg and Russell as previously described.13 Briefly, tissue extract ali- Animals quots (10 ml) in triplicate were incubated in buffer (75 mM glycine, pH 9.0, 0.25 mM MnCl2) containing 14 300 000 dpm of [ C-guanidino]L-arginine (51.5 mCi/ All studies were approved by the Institutional mmol; NEN Life Science Products, Boston, MA, Animal Care and Use Committee at Boston Uni- USA), 4 mM of unlabelled L-arginine in a final versity School of Medicine. Female New Zealand volume of 100 ml. Samples were incubated for White rabbits (4.0–4.5 kg) were anesthetized with 60 min at 371C and reactions were terminated by intramuscular injections of ketamine (35 mg/kg) and the addition of 400 ml of 0.25 M acetic acid (pH 4.5), xylazine (5 mg/kg). For in vivo studies, anesthesia 7 M urea, 10 mM L-arginine. After the addition of was maintained with intravenous 50 mg/ml sodium 500 ml of water, samples were passed through a nembutal (0.2 ml PRN). For tissue collection, ani- 0.5 ml column of Dowex 50W-X8 resin (Bio-Rad mals were euthanized with 60 mg/kg of sodium Laboratories, Hercules, CA, USA). Tubes were nembutal. Vaginal tissue was removed, separated rinsed twice with 500 ml of water and both rinses into proximal and distal portions and frozen in were loaded onto the columns. Columns were liquid nitrogen for assays. washed with 1 ml of water and all effluent was collected in 20 ml vials. After the addition of 16 ml of Liquiscint (National Diagnostics, Atlanta, GA, Preparation of vaginal tissue extracts USA), radioactivity was quantified by liquid scin- tillation spectroscopy. Urea production was normal- Frozen vaginal tissues were pulverized and the ized to total soluble protein in the tissue. resulting tissue powder was homogenized (1 g/3 ml) in ice-cold 20 mM HEPES (pH 7.20) containing 250 mM sucrose, 1 mM EDTA and protease inhibitor Pelvic nerve stimulation cocktail (1 mM AEBSF, 0.8 mM aprotinin, 20 mM leupeptin, 40 mM bestatin, 15 mM pepstatin A and Anesthetized rabbits were secured in the supine 14 mM E-64; catalog #P8340; Sigma, St Louis, MO, USA). The total cell extract was centrifuged at position and a 3 cm midline neck incision was 1000 g for 30 min at 41C. The supernatant was fashioned to access the carotid artery. A 20-guage removed and either used immediately or stored angiocatheter was inserted into the carotid artery frozen at À751C until experimentation. and connected to a PT300 pressure transducer (Grass Instruments/Astro-Med, Inc., Warwick, RI, USA) to monitor systemic blood pressure continu- ously. A 4 cm lower midline abdominal incision was Determination of NOS activity fashioned to expose the pelvic nerve. A bipolar platinum wire electrode was carefully positioned on NOS activity in the total tissue extract was deter- the isolated pelvic nerve to avoid nerve injury. 3 mined by conversion of L-[2,3- H]arginine (NEN Life Unilateral pelvic nerve stimulation (PNS) was Science Products, Boston, MA, USA) to [3H]citrul- accomplished with a Grass S9 stimulator set at line and NO, as previously described.14 Briefly, normal polarity and repeat mode to generate a 30 s aliquots of the tissue extract were incubated with train of square waves with 10 V pulse amplitude and (3 mM), (30 U/ml), L- 0.8 msec pulse duration. For each animal, a stimula- 3 arginine (50 mM), L-[ H]arginine (2 mCi/ml), NADPH tion frequency causing submaximal response was (20 mM) and CaCl2 (1 mM)at371C for 45 min. Parallel selected as the lowest frequency that demonstrated samples were incubated at 21C to determine non- two consecutive reproducible control responses. specific activity. Citrulline was separated from Based on our previous experience, stimulation arginine by passing samples through 1 ml columns frequencies below 8 Hz result in submaximal re- of AG50W-X8 resin (Bio-Rad Laboratories, Hercules, sponses and 2 or 4 Hz stimulations were typically CA, USA) and quantified by scintillation spectro- adequate. Hemodynamic parameters were continu- scopy. Citrulline production was normalized to total ously recorded throughout the experiment. soluble protein in the tissue.

Genital blood flow Determination of arginase activity A dual channel laser oximeter (Model 96208; ISS, Crude tissue extracts were derived from homoge- Inc., Champaign, IL, USA) was utilized to measure nates of rabbit vaginal tissue. Arginase enzyme genital blood flow, as described previously.15 The skin

International Journal of Impotence Research NO-cGMP in regulation of vaginal blood flow SW Kim et al 357 around the labia was carefully shaved to ensure Results good contact with the optical fibers. The probe was positioned longitudinally, externally over the cli- toris, labia and lower vagina such that the detector Biochemical studies fiber was positioned just below the pubic arch. Changes in blood flow were detected as changes in the concentration of oxyhemoglobin. Activity for NOS and arginase was detected in both the proximal and distal vagina. As shown in Figure 1a, citrulline production, indicative of NOS activity, was not significantly different in the Vaginal blood flow proximal vagina (513.27153.7 pmol/mg prot.) com- pared to the distal vagina (360.5782.4 pmol/mg Vaginal blood flow was assessed by laser Doppler prot.). On the other hand, arginase activity flowmetry (Model BLF21D, Transonic System Inc, (Figure 1b) was significantly higher (Po0.05) in 7 Ithaca, NY, USA). A laser Doppler surface probe the distal vagina (198.6 6.1 nmol urea/mg prot.) 7 (length 14 mm, width 6 mm, height 3 mm) was vs the proximal vagina (58.3 1.4 nmol urea/mg placed directly in the vaginal canal. The probe prot.). was faced toward the lateral or posterior vaginal wall. The data were expressed in tissue perfusion units.

In vivo study protocol

Responses to PNS were measured using either oximetry (for genital blood flow) or laser Doppler flowmetry (for vaginal blood flow). Control re- sponses to PNS were determined in each animal. Vehicle (40% propylene glycol or DMSO) was administered intravenously and PNS was repeated after 10 min. Upon return to baseline values, animals then received intravenous administration G of 60 mg/kg of the NOS inhibitor N -nitro-L-arginine methyl ester (L-NAME) in saline, 6 mg/kg of the arginase inhibitor 2(S)-amino-6-boronohexanoic acid (ABH) in 40% propylene glycol, 1 mg/kg of the guanylate cyclase inhibitor 1H-[1,2,4]oxadiazo- lo[4,3,-a]quinoxalin-1-one (ODQ) in DMSO or 0.2 mg/kg of the PDE 5 inhibitor sildenafil citrate in saline. PNS was repeated 10 min after adminis- tration of a given .

Data analysis

For all laser oximetry and Doppler flowmetry recordings, the area under the curve (AUC) for each response was determined using Microsoft Excel or Windaq (Dataq Instruments, Akron, OH, USA) software. All in vivo responses obtained in the presence of each enzyme inhibitor were transformed as percent of control and the results were expressed as mean7s.d. (n ¼ 5, unless other- wise specified). Data were analyzed by the paired Figure 1 NOS and arginase enzyme activity in rabbit vagina. (a) t-test or one-way ANOVA followed by the Tukey– NOS enzyme activity was assessed in tissue extracts from proximal and distal rabbit vagina by measuring the conversion Kramer post hoc test. Means were considered to be of [3H]arginine to [3H]citrulline. (b) Arginase enzyme activity was significantly different for P-values less than or equal determined by measuring conversion of [14C]arginine to [14C]urea. to 0.05. Data are mean7s.e.m. (*Po0.05).

International Journal of Impotence Research NO-cGMP in regulation of vaginal blood flow SW Kim et al 358 In vivo studies

PNS caused significant increases in genital tissue oxyhemoglobin concentration and vaginal blood flow. Repeated PNS with 10 min rest intervals did not result in significant changes in the genital oxyhemoglobin and vaginal blood flow responses. Also, baseline values of genital oxyhemoglobin and vaginal blood flow did not vary significantly after intravenous injection of L-NAME, ABH, ODQ or sildenafil at the doses administered in this study. L-NAME significantly suppressed responses to PNS by 66.3% for genital oxyhemoglobin and by 52.8% for vaginal blood flow 10 min after admi- nistration (Figure 2). As shown in Figure 3, ABH

Figure 3 Effects of arginase inhibitor ABH on genital oxyhemo- globin concentration and vaginal blood flow. Changes in oxyhemoglobin (a) and vaginal blood flow (b) in female rabbits in response to PNS at the submaximal frequency were assessed by laser oximetry and laser Doppler flowmetry, respectively. All data were expressed as relative changes of AUC for genital tissue oxyhemoglobin (P ¼ 0.07 relative to control response; n ¼ 3) and vaginal blood flow (*Po0.05, relative to control response; n ¼ 5).

administration increased PNS-induced changes in genital tissue oxyhemoglobin and vaginal blood flow. However, the increase in oxyhemoglobin did not reach statistical significance when compared to control or vehicle (P ¼ 0.07), whereas the increase in vaginal blood flow was statistically significant (Po0.05). ABH administration had no discernible effect on systemic arterial blood pressure (data not shown). Figure 2 Effects of NOS inhibitor L-NAME on genital oxyhe- However, intravenous injection of L-NAME caused moglobin concentration and vaginal blood flow. Changes in an increase in mean arterial blood pressure. As oxyhemoglobin (a) and vaginal blood flow (b) in female rabbits shown in Figure 4, the mean arterial blood pressure in response to PNS at submaximal frequency were assessed by 7 laser oximetry and laser Doppler flowmetry, respectively. All data 10 min after L-NAME injection (81.6 7.8 mmHg) were expressed as relative changes of AUC (area under the curve). was significantly higher (Po0.05) than baseline (*Po0.05, relative to control response; n ¼ 5). (59.677.6 mmHg). The increase in blood pressure

International Journal of Impotence Research NO-cGMP in regulation of vaginal blood flow SW Kim et al 359

Figure 6 Effect of PDE 5 inhibitor sildenafil on vaginal blood flow. Changes in vaginal blood flow in female rabbits in response to PNS at the submaximal frequency were assessed by laser Doppler flowmetry. Data were expressed as relative changes of AUC. (*Po0.05, relative to control response; n ¼ 5).

ODQ (Figure 5) significantly inhibited PNS-induced vaginal blood flow by 36%, while systemic admin- Figure 4 Mean arterial blood pressure before and after L-NAME istration of sildenafil (Figure 6) significantly en- injection. (a) Representative pressure recordings of changes in mean arterial blood pressure. (b) Mean arterial blood pressure was hanced PNS-induced vaginal blood flow by 44%. significantly increased after 10 min of intravenous L-NAME Intravenous administration of ODQ showed no administration (*Po0.05; n ¼ 5). effect on systemic arterial blood pressure. On the other hand, intravenous injection of sildenafil caused a mild, transient drop in systemic blood pressure, which recovered to baseline values within 10 min of the injection at which time PNS was repeated.


The importance of the NO-cGMP pathway in mediating neurogenic relaxation of clitoral caverno- sal smooth muscle has been demonstrated.9,16 In contrast, the role of this pathway in the vagina has only been partially investigated. Using tissue strips in organ bath preparations, Ziessen et al11 charac- terized NANC neurogenic relaxation of the rabbit vaginal muscularis (nonvascular smooth muscle). In Figure 5 Effect of guanylate cyclase inhibitor ODQ on vaginal this study, Ziessen et al observed that the degree of blood flow. Changes in vaginal blood flow in female rabbits in inhibition attained with L-NAME (an NOS inhibitor) response to PNS at the submaximal frequency were assessed by or ODQ (guanylate cyclase inhibitor) was no greater laser Doppler flowmetry. Data were expressed as relative changes * than 30%, suggesting that a non-nitrergic compo- of AUC ( Po0.05, relative to control response; n ¼ 5). nent is the major mediator of NANC relaxation response in the rabbit vagina. Interestingly, this non- nitrergic NANC response was not associated with caused by L-NAME was rapid in onset, reaching a any known neuropeptides or purines and the main plateau within 5–10 min and was sustained for more relaxatory neurotransmitter remains unidentified.11 than 1 h after administration. On the other hand, in a separate study using rat The effects of ODQ and sildenafil were only distal vaginal tissue, Giraldi et al10 completely assessed by laser Doppler flowmetry. Intravenous inhibited NANC relaxation using inhibitors of the administration of the guanylate cyclase inhibitor NO-cGMP pathway. Thus, the NO-cGMP pathway

International Journal of Impotence Research NO-cGMP in regulation of vaginal blood flow SW Kim et al 360 appears to exert differential regulation of smooth intracellular arginine stores by arginase. However, muscle function throughout the female genital tract this indirect mechanism of NOS regulation may and its predominance in the vagina may be species - have other consequences in terms of host defense specific. responses.19,20 In other tissues, arginase (via argi- To date, there have been no functional studies nine ) has been shown to participate in examining the role of NO in regulating vaginal blood inflammation and wound healing.20 In addition, flow. In our study, the inhibition of NOS activity in arginase can regulate the biosynthesis of polya- vivo by L-NAME resulted in a significant reduction mines, which are critical for cell growth and in genital tissue oxyhemoglobin and vaginal blood proliferation.20 The presence of higher arginase flow. Further, the guanylate cyclase inhibitor ODQ activity in the distal vagina may be important for significantly reduced PNS-induced vaginal blood some or all of these processes in addition to flow, while sildenafil increased vaginal blood flow. regulation of smooth muscle tone. Nevertheless, These findings are consistent with our previous the presence of both NOS and arginase within the studies on the facilitating effects of sildenafil on same tissue in conjunction with our hemodynamic sexual arousal response measured by oximetry, data suggest that these two enzymes may regulate vaginal luminal pressure and vaginal lubrication.15 the production of NO in the vagina in a reciprocal The inhibitory effect of L-NAME was somewhat fashion. greater when assessed by genital tissue oxyhemo- Another consideration regarding the differential globin (66.3% inhibition) than vaginal blood flow distribution of enzymes is that the vagina is derived (52.8% inhibition). Although this difference was not from different embryological structures during de- statistically significant, the difference in mean velopment. The distal third of the human vagina is percent inhibition may be due to the fact that laser derived from the urogenital sinus and the proximal oximetry measures oxyhemoglobin concentration in two thirds from fused mullerian ducts. Moreover, an both vaginal and clitoral tissues, whereas laser immunohistochemical study of normal human va- Doppler flowmetry measures blood flow only in ginal tissues demonstrated more nerve fibers in the the vagina. The inhibition of vaginal blood flow distal vagina compared to the proximal vagina.21 occurred in spite of the rise in mean arterial blood Therefore, differences in enzyme distribution be- pressure after L-NAME administration. This rise in tween the proximal and distal portions of the vagina systemic blood pressure is consistent with other may be reflective of differences in development as studies that have investigated the role of NOS well as differences in function. inhibitors on the regulation of blood pressure and Future in vivo studies investigating hemodynamic is most likely due to the inhibition of NOS in the regulation in vaginal tissue will need to consider the endothelium of the resistance arteries.17,18 most appropriate method to quantify engorgement The inhibition of arginase activity by ABH caused responses, as well as the potential differences an enhancement of genital and vaginal blood flow. between the proximal and distal regions of the These effects of ABH might be due to an increase in vagina. We quantified the effects of each inhibitor by the availability of substrate for NOS similar to that comparison of the AUC for each response, as seen in penile corpus cavernosum.12,13 On average, opposed to comparing peak responses. While oxi- the effect of ABH was greater on genital tissue metry can provide more stable recordings, measure- oxyhemoglobin than on vaginal blood flow. As ment of peak responses for laser Doppler flowmetry mentioned above, this discrepancy may be due to is problematic for several reasons. Using laser differences between the two techniques. It is inter- Doppler flowmetry, recordings of flow rate in the esting to note that arginase enzyme activity was vagina during pelvic nerve stimulation exhibit an greater in the distal vagina than in the proximal immediate increase with sometimes rapid fluctua- vagina, while the distribution of NOS was similar tions. Since some of these fluctuations are due to throughout the vagina. Although we did not char- transient vaginal contractions, it is difficult to acterize which enzyme subtypes were present and accurately determine the true peak blood flow did not examine the cellular distribution of NOS during the stimulation period. In addition, peak and arginase in the rabbit vagina, recent studies by blood flows that occur for only a short duration Giraldi et al10 demonstrated NOS immunoreactivity provide little information regarding the overall in plexuses of nerve terminals surrounding vaginal quality of the engorgement response. Lastly, the rate arteries, as well as in a localized region of circular of flow can only increase to a finite level that is smooth muscle of the distal rat vagina. In future determined by the arterial-venous pressure gradient. studies, it may be important to investigate regional In contrast, AUC reflects a measure of perfusion variations in vaginal blood flow. integrated over time and consequently incorporates The role of arginase in genital tissues has not been ‘duration of response’ data. Thus, AUC may be a well defined. Demonstration of its functional sig- better parameter to measure the overall engorgement nificance has been limited to its inhibitory effect on response in the vagina. This is consistent with the NO-mediated relaxation in penile corpus caverno- known mechanism of vaginal engorgement. While sum.12,13 This has been attributed to lowering of there may be partial vasoconstriction in the veins

International Journal of Impotence Research NO-cGMP in regulation of vaginal blood flow SW Kim et al 361 during arousal, there is no veno-occlusive mechan- 5 Ottesen B et al. Vasoactive intestinal polypeptide and the ism in the vagina. Thus, engorgement is achieved by female genital tract: relationship to reproductive phase and a sustained period of increased arterial flow that delivery. Am J Obstet Gynecol 1982; 143: 414 – 420. 6 Ottesen B. Vasoactive intestinal polypeptide as a neurotrans- overcomes venous drainage. This assumption ne- mitter in the female genital tract. Am J Obstet Gynecol 1983; cessitates the inclusion of the time dimension in 147: 208 – 224. measuring vaginal engorgement. 7 Ottesen B et al. Vasoactive intestinal polypeptide (VIP) In conclusion, our results demonstrate that in- provokes vaginal lubrication in normal women. Peptides 1987; 8: 797 – 800. hibition of individual enzymes in the NO-cGMP 8 Palle C, Bredkjaer HE, Ottesen B, Fahrenkrug J. Vasoactive pathway results in consistent and significant altera- intestinal polypeptide and human vaginal blood flow: com- tions in vaginal hemodynamics and suggest that parison between transvaginal and intravenous administration. this pathway is an important regulatory mechanism Clin Exp Pharmacol Physiol 1990; 17: 61 – 68. of vaginal blood flow during sexual arousal. Further, 9 Cellek S, Moncada S. Nitrergic neurotransmission mediates the non-adrenergic non-cholinergic responses in the clitoral we report the first in vivo evidence of a physio- corpus cavernosum of the rabbit. Br J Pharmacol 1998; 125: logical role for arginase in the regulation of NO 1627 – 1629. production within the vagina. 10 Giraldi A et al. Morphological and functional characterization of a rat vaginal smooth muscle sphincter. Int J Impot Res 2002; 14: 271 – 282. 11 Ziessen T, Moncada S, Cellek S. Characterization of the non- nitrergic NANC relaxation responses in the rabbit vaginal Acknowledgements wall. Br J Pharmacol 2002; 135: 546 – 554. 12 Cox JD, Kim NN, Traish AM, Christianson DW. Arginase– boronic acid complex highlights a physiological role in This work was supported by Grants R01-DK 56846 erectile function. Nat Struct Biol 1999; 6: 1043 – 1047. 13 Kim NN et al. Probing erectile function: S-(2-boronoethyl)-L- and K01-DK02696 from the National Institute of cysteine binds to arginase as a transition state analogue and Diabetes and Digestive and Kidney Diseases. We enhances smooth muscle relaxation in human penile corpus thank Drs David W Christianson and Hyunshun cavernosum. Biochemistry 2001; 40: 2678 – 2688. Shin (Department of Chemistry, University of Penn- 14 Kim N et al. Oxygen tension regulates the nitric oxide sylvania) for synthesizing and providing the argi- pathway. Physiological role in penile erection. J Clin Invest 1993; 91: 437 – 442. nase inhibitor ABH and Jerie McGrath-Cerqua for 15 Min K et al. Sildenafil augments pelvic nerve-mediated female her administrative assistance. genital sexual arousal in the anesthetized rabbit. Int J Impot Res 2000; 12(Suppl 3): S32 – S39. 16 Vemulapalli S, Kurowski S. Sildenafil relaxes rabbit clitoral corpus cavernosum. Life Sci 2000; 67: 23 – 29. 17 Rees DD, Palmer RM, Moncada S. Role of endothelium- References derived nitric oxide in the regulation of blood pressure. Proc Natl Acad Sci USA 1989; 86: 3375 – 3378. G 18 Schwarzacher S, Raberger G. L-N -nitro-arginine methyl ester 1 Levin RJ. Sex and the human female reproductive tractFwhat in the anesthetized rabbit: venous vasomotion and plasma really happens during and after coitus. Int J Impot Res 1998; levels. J Vasc Res 1992; 29: 290 – 292. 10(Suppl 1): S14 – S21. 19 Efron DT, Barbul A. Modulation of inflammation and 2 Wagner G. Aspects of genital physiology and pathology. Semin immunity by arginine supplements. Curr Opin Clin Nutr Neurol 1992; 12: 87 – 97. Metab Care 1998; 1: 531 – 538. 3 Hoyle CH et al. Innervations of vasculature and microvascu- 20 Wu G, Morris SM. Arginine metabolism: nitric oxide and lature of the human vagina by NOS and neuropeptide- beyond. Biochem J 1998; 336: 1 – 17. containing nerves. J Anat 1996; 188: 633 – 644. 21 Hilliges M, Falconer C, Ekman-Ordeberg G, Johansson O. 4 Levin RJ. VIP, vagina, clitoral and periurethral glansFan Innervation of the human vaginal mucosa as revealed by update on human female genital arousal. Exp Clin Endocrinol PGP 9.5 immunohistochemistry. Acta Anat (Basel) 1995; 153: 1998; 198: 61 – 69. 119 – 126.

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